Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity

ABSTRACT

The application is related to compositions and methods for the treatment of hypersensitivity, wherein the compositions comprise a particle packaged with immunostimulatory nucleic acids. The compositions of the invention are particularly useful in the treatment of atopic eczema, asthma and IgE-mediated allergy (type I allergy), especially pollen allergy and house dust allergy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/638,664, filed Dec. 14, 2006, and which claims the benefit of U.S.Provisional Application No. 60/750,042, filed Dec. 14, 2005, and U.S.Provisional Application No. 60/812,592, filed Jun. 12, 2006, thedisclosures of each of which are entirely incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

This invention relates to the field of immunology and the treatment ofhypersensitivity by immunologically active compositions. Thecompositions of the invention comprise particles, preferably virus-likeparticles, nanoparticles, microparticles or liposomes which are packagedwith an immunostimulatory nucleic acid. The compositions of theinvention are useful in the treatment of hypersensitivity, preferablyallergy, including diseases such as atopic eczema, asthma andIgE-mediated allergy (type-I allergy), especially pollen allergy (hayfever). The invention therefore also provides methods for the treatmentof these diseases.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a composition for use as amedicament, the composition comprising, essentially consisting of, orconsisting of a particle and an immunostimulatory nucleic acid (ISS-NA),wherein said particle is packaged with said ISS-NA.

It was surprisingly found that the inventive compositions are useful inthe prophylactic and therapeutic treatment of hypersensitivity,preferably allergy. In a further aspect the invention thus provides acomposition for use in a method of treating hypersensitivity, preferablyallergy in an animal, the composition comprising a particle andimmunostimulatory nucleic acid (ISS-NA), wherein said particle ispackaged with said ISS-NA. In a specific aspect the invention provides acomposition for use in a method of treating hypersensitivity, preferablyallergy in an animal, the composition comprising a virus-like particleand an unmethylated CpG-containing oligonucleotide, wherein saidvirus-like particle is packaged with said unmethylated CpG-containingoligonucleotide. In a preferred embodiment said hypersensitivity is anallergy, preferably IgE-mediated asthma, atopic eczema or IgE-mediatedallergy (type I allergy). In a further preferred embodiment saidparticle is selected from a nanoparticle, a microparticle and aliposome. In a further preferred embodiment said particle is a VLP,preferably a VLP of an RNA-bacteriophage, again preferably a VLP ofbacteriophage Qβ. In a further preferred embodiment said unmethylatedCpG-containing oligonucleotide exclusively consists of phosphodiesterbound nucleotides, most preferably of G10 (SEQ ID NO:27).

In a further aspect the invention provides a process of producing acomposition for use in a method of treating hypersensitivity in ananimal, said composition comprising (a) a virus-like particle; and (b)an unmethylated CpG-containing oligonucleotide; wherein said virus-likeparticle (a) is packaged with said unmethylated CpG-containingoligonucleotide (b), said process comprising the steps of (i) incubatingsaid VLP (a) with said unmethylated CpG-containing oligonucleotide (b);(ii) adding RNase; and (iii) purifying said composition.

In a further aspect, the invention provides a process of producing acomposition for use in a method of treating hypersensitivity in ananimal, said composition comprising (a) a virus-like particle; and (b)an unmethylated CpG-containing oligonucleotide; wherein said virus-likeparticle (a) is packaged with said unmethylated CpG-containingoligonucleotide (b), said process comprises the steps of (i) incubatingsaid VLP with RNase; (ii) adding said unmethylated CpG-containingoligonucleotide; and (iii) purifying the composition.

In a further aspect the invention provides a process of producing acomposition for use in a method of treating hypersensitivity in ananimal, said composition comprising (a) a virus-like particle; and (b)an unmethylated CpG-containing oligonucleotide; wherein said virus-likeparticle (a) is packaged with said unmethylated CpG-containingoligonucleotide (b) said process comprising the steps of (i)disassembling said VLP; (ii) adding said unmethylated CpG-containingoligonucleotide; and (iii) reassembling said VLP.

In a further aspect the invention provides a process of producing acomposition for use in a method of treating hypersensitivity in ananimal, said composition comprising (a) a virus-like particle; and (b)an unmethylated CpG-containing oligonucleotide; wherein said virus-likeparticle (a) is packaged with said unmethylated CpG-containingoligonucleotide (b) said process comprises the steps of (i) incubatingsaid VLP with solutions comprising metal ions capable of hydrolyzing thenucleic acids of said VLP; (ii) adding said unmethylated CpG-containingoligonucleotide; and (iii) purifying said composition, whereinpreferably said metal ions of step (i) are selected from the groupconsisting of (a) zinc (Zn) ions; (b) copper (Cu) ions; (c) iron (Fe)ions; (d) any mixtures of at least one ion of (a), (b) and/or (c). In apreferred embodiment, said VLP is produced in a bacterial expressionsystem.

In a further aspect the invention provides a process of producing acomposition for use in a method of treating hypersensitivity in ananimal, said composition comprising (a) a virus-like particle; and (b)an unmethylated CpG-containing oligonucleotide; wherein said virus-likeparticle (a) is packaged with said unmethylated CpG-containingoligonucleotide (b) said process comprises the steps of (i) incubatingsaid VLP under alkaline conditions, preferably in the presence of NaOH,most preferably in the presence of about 25 mM NaOH; (ii) adding saidunmethylated CpG-containing oligonucleotide; and (iii) purifying saidcomposition.

In a further aspect the invention provides a process of producing acomposition for use in a method of treating hypersensitivity in ananimal, said composition comprising (a) a virus-like particle; and (b)an unmethylated CpG-containing oligonucleotide; wherein said virus-likeparticle (a) is packaged with said unmethylated CpG-containingoligonucleotide (b), said process comprising the steps of (i) incubatingsaid VLP with a solution capable of destabilizing said VLP, whereinpreferably said VLP is a VLP of bacteriophage Qβ; (ii) purifying thecoat protein from said solution; and (iii) reassembling said coatprotein to a VLP in the presence of unmethylated CpG-containingoligonucleotide and an oxidizing agent.

In a further aspect the invention provides compositions for use as amedicament, wherein said compositions are obtainable by a processcomprising the steps of (i) incubating a VLP with unmethylatedCpG-containing oligonucleotides; (ii) adding RNase; and (iii) purifyingsaid composition.

In a further aspect the invention provides compositions for use as amedicament, wherein said compositions are obtainable by a processcomprising the steps of (i) incubating a VLP with RNase; (ii) addingunmethylated CpG-containing oligonucleotides; and (iii) purifying thecomposition.

In a further aspect the invention provides compositions for use as amedicament, wherein said compositions are obtainable by a processcomprising the steps of (i) disassembling a VLP: (ii) addingunmethylated CpG-containing oligonucleotides; and (iii) reassemblingsaid VLP.

In a further aspect the invention provides compositions for use as amedicament, wherein said compositions are obtainable by a processcomprising the steps of (i) incubating a VLP with solutions comprisingmetal ions capable of hydrolyzing the nucleic acids of said VLP; (ii)adding unmethylated CpG-containing oligonucleotides; and (iii) purifyingsaid composition.

In a further aspect the invention provides compositions for use in amethod of treating allergy in an animal, wherein said compositions areobtainable by a process comprising the steps of (i) incubating a VLPwith unmethylated CpG-containing oligonucleotides; (ii) adding RNase;and (iii) purifying said composition.

In a further aspect the invention provides compositions for use in amethod of treating allergy in an animal, wherein said compositions areobtainable by a process comprising the steps of (i) incubating a VLPwith RNase: (ii) adding unmethylated CpG-containing oligonucleotides;and (iii) purifying the composition.

In a further aspect the invention provides compositions for use in amethod of treating allergy in an animal, wherein said compositions areobtainable by a process comprising the steps of (i) disassembling a VLP;(ii) adding unmethylated CpG-containing oligonucleotides; and (iii)reassembling said VLP.

In a further aspect the invention provides compositions for use in amethod of treating allergy in an animal, wherein said compositions areobtainable by a process comprising the steps of (i) incubating a VLPwith solutions comprising metal ions capable of hydrolyzing the nucleicacids of said VLP; (ii) adding unmethylated CpG-containingoligonucleotides; and (iii) purifying said composition.

In a further aspect the invention provides pharmaceutical compositionscomprising a composition of the invention.

In a further aspect the invention provides a method of treatinghypersensitivity, preferably allergy in an animal, said methodcomprising introducing into said animal a composition of the invention.

A further aspect of the invention is the use of a composition of theinvention or of a pharmaceutical composition of the invention for themanufacture of a pharmaceutical for the treatment of hypersensitivity inan animal, wherein preferably said hypersensitivity is an allergy,wherein further preferably said allergy is selected from the groupconsisting of: (a) IgE-mediated asthma, (b) atopic eczema; and (c)IgE-mediated allergy (type I allergy), preferably pollen allergy (hayfever) or house dust allergy.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1: Characterization of purified Q§coat protein by analytical sizeexclusion chromatography. (A) sample of purified Qβ VLP. The observedpeak (ratio A260/A280=2) is dominated by the RNA core of the VLP,because the absorption coefficient of RNA at 260 nm is approx. 100 foldhigher than the absorption coefficient of the coat protein. (B) sampleof the supernatant of the disassembly reaction. Released coat protein isindicated by the presence of the protein-like peak at approx. 12 min.Furthermore several species of non-precipitated RNA molecules arepresent in the range 6.8 to 11 min. (C) sample of purified Qβ coatprotein. Analysis was performed in PBS on column TSK G5000 PW×1 (TosohBioscience).

FIG. 2: Analytical size exclusion chromatography of (A) native Qβ VLP,(D) QβG10 VLP and the packaging components (B) oligo nucleotide G10 and(C) Qβ coat protein. The observed peak for QβG10 VLP (D) (ratioA260/A280=1.74) is dominated by the G10 core of the VLP, because theabsorption coefficient of G10 at 260 nm is approx. 130 fold higher thanthe absorption coefficient of the coat protein. Analysis was performedin PBS on column TSK G5000 PW×1 (Tosoh Bioscience).

FIG. 3: Non-reducing SDS-PAGE analysis of native Qβ VLP and in vitroassembled QβG10. The position of the coat protein pentamers and hexamersis indicated ((a) molecular weight marker, (b) Qβ VLP, (c) Qβ G10).

FIG. 4: Ratio of the number of VLP+ cells at 2 h over the number of VLP+cells at 24 h in the myeloid-DC, lymphoid-DC, Macrophage, pDC and B-cellpopulations after subcutaneous injection in the footpad, as a measure ofDC-mediated or free draining of VLP to the Lymphnode. Anti-CD11c,-CD11b, -B220 and -CD19 antibodies were used to identify myeloid- andlymphoid-DC, Macrophages, pDCs and B cells by FACS analysis.

FIG. 5: Ratio between percentages of particle-positive DC andmacrophages. Mice were injected with nanoparticles of different size. 48h later cells were analyzed by FACS.

FIG. 6: Activation of BMDCs by QβG10. BMDCs were activated by QβG10 andhence secreted IL-12 in a dose dependent manner (dose is given asequivalent of G10 oligonucleotide packaged in the Qβ VLPs) whileuntreated control cells did not secrete any IL-12.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs.

“hypersensitivity”: In the context of the invention the term“hypersensitivity” is to be understood as suggested in Johansson et al.2001, Allergy 56:813-824 as any objectively reproducible symptom orsigns, initiated by exposure to a defined stimulus at a dose toleratedby normal subjects. Hypersensitivity reactions are reproducible in thesense that there is reasonable evidence from history, examination, orinvestigation of a link between the symptoms and the environmentalfactors to which the patients attribute their symptoms. The termhypersensitivity encompasses non-allergic hypersensitivity and allergichypersensitivity (allergy). Non-allergic hypersensitivity is anyhypersensitivity which does not comprise an involvement of the immunesystem or at least where no involvement of the immune system can bedetected, wherein allergic hypersensitivity always comprises aninvolvement of the cellular or humoral immune system. Hypersensitivitypreferably refers to allergic and non-allergic forms of a diseaseselected from the group consisting of: (a) asthma, (b) rhinitis, (c)conjunctivitis, (d) rhinoconjuctivitis, (e) dermatitis, (e) urticaria,(c) food hypersensitivity, (c) drug hypersensitivity, (c) inacct stingor bite hypersensitivity, and (e) anaphylaxis. In particular,hypersensitivity also includes any type of allergy.

Further preferably, hypersensitivity refers to allergic forms of adisease selected from the group consisting of: (a) asthma, (b) rhinitis,(c) conjunctivitis, (d) rhinoconjuctivitis, (e) dermatitis, (e)urticaria, (e) food hypersensitivity, (e) drug hypersensitivity, (e)insect sting or bite hypersensitivity, and (e) anaphylaxis. Inparticular, hypersensitivity also includes any type of allergy.

“allergy”: In the context of the application the term “allergy” standsfor “allergic hypersensitivity” and is to be understood as suggested byJohansson et al. 2001, Allergy 56:813-824 and Johannson et al. 2004, J.Allergy Clin. Immunol. 113(5) 832-835. Unless otherwise indicated, theapplication follows the nomenclature for allergy as set forth therein.Allergy or allergic hypersensitivity is a hypersensitivity reactioninitiated by immunologic mechanisms in response to a substance(allergen), often in a genetically predisposed individual (atopy).Allergy can be antibody- or cell-mediated. In most patients, theantibody typically responsible for an allergic reaction belongs to theIgE isotype (see “antibodies”) and these patients may be said to sufferfrom IgE-mediated allergy (type-I allergy). It must be noted that notall IgE-mediated allergic reactions occur in atopic subjects. In nonIgE-mediated allergy, the antibody may belong to the IgG isotype. Thus,within the meaning of the application, “allergy” refers to both,IgE-mediated allergy (type-I allergy) and non IgE-mediated allergy.IgE-mediated allergy is preferably addressed by the invention.Therefore, in the context of the invention allergy preferably refers toIgE-mediated allergy. Allergies are classified according to the sourceof the antigen evoking the hypersensitive reaction. In one embodimentallergy is selected from (a) food allergy, (b) drug allergy, (c) housedust allergy, (d) insect venom or bite allergy, and (e) pollen allergy.Alternatively, allergies are classified based on the major symptoms ofthe hypersensitive reaction. Thus, in another embodiment allergy refersto any allergic form of a disease selected from the group of (a) asthma,(b) rhinitis, (c) conjunctivitis, (d) rhinoconjuctivitis, (e)dermatitis, (f) urticaria and (g) anaphylaxis.

“type-I allergy”: the terms “type-I allergy” and “IgE-mediated allergy”are used interchangeably and relate to IgE-mediated hypersensitivitiesto allergens. Preferred embodiments of the invention relate toIgE-mediated allergy selected from the group consisting of (a) pollenallergy (hay fever); (b) house dust allergy; (c) food allergy; (d) drugallergy; (e) insect venom or bite allergy, preferably bee venom allergy;and (f) animal allergy, preferably cat allergy.

“hay fever”: typical form of an IgE-mediated allergy (type-I allergy)against pollen which may comprise rhinitis, conjunctivitis and/orasthma, wherein asthma preferably occurs in chronic forms of hay fever.

“atopy”, “atopic diseases”: Atopy is a personal or familial tendency toproduce IgE antibodies in response to low doses of allergens, usuallyproteins, and to develop typical symptoms such as asthma,rhinoconjunctivitis, or eczema/dermatitis. The first manifestations ofatopy in a child are often allergic symptoms, such as diarrhea,wheezing, and skin rashes, and only later can the responsible IgEantibody be detected. Allergic symptoms in a typical atopic individualmay be referred to as atopic. In one embodiment of the inventionhypersensitivity is an atopic disease, preferably an atopic diseaseselected from the group consisting of (a) atopic asthma, (b) atopiceczema, (c) atopic IgE-mediated allergy, preferably pollen allergy (hayfever), house dust allergy or house dust mite allergy. In one embodimentthe application relates to IgE-mediated allergy in general, irrespectiveof whether or not said IgE-mediated allergy is regarded as atopic or nonatopic allergy. However, specifically preferred embodiments of theinvention relate to atopic allergy, preferably to IgE-mediated atopicallergy.

“rhinitis”: The term “rhinitis” relates to hypersensitivity symptomsfrom the nose, for example, itching, sneezing, increased secretion, andblockage. Rhinitis relates to non-allergic as well as allergic, i.e.immunologically mediated, rhinitis. Preferred embodiments of theinvention relate to allergic rhinitis, preferably to IgE-mediated andnon IgE-mediated forms of allergic rhinitis. Specifically preferredembodiments relate to IgE-mediated allergic rhinitis.

“conjunctivitis”: The term conjunctivitis relates to irritations of theeye which can be of allergic as well as non-allergic origin, whereinallergic conjunctivitis encompasses IgE-mediated and non IgE-mediatedallergic conjunctivitis. Allergic conjunctivitis, especially IgEmediated allergic conjunctivitis is commonly accompanied by allergicrhinitis, so this disorder is appropriately termed allergicrhinoconjuctivitis. Besides IgE-mediated conjunctivitis, contactallergic conjunctivitis involving TH1 mechanisms occurs. Non-allergicconjunctivitis also often accompanies non-allergic rhinitis. Preferredembodiments of the invention relate to allergic conjunctivitis,including IgE-mediated and non IgE-mediated forms of allergicconjunctivitis. Specifically preferred embodiments relate toIgE-mediated allergic conjunctivitis. Further preferred embodimentsrelate to IgE-mediated allergic rhinoconjunctivitis.

“asthma”: Asthma or asthma bronchiale is a chronic respiratory diseasedue to inflammation of the air passages in the lungs and affects thesensitivity of the nerve endings in the airways so they become easilyirritated. In an attack, the lining of the passages swell causing theairways to narrow and reducing the flow of air in and out of the lungs.Asthma can occur in a intermittent form (2 attacks per week or lessduring daytime, 2 attacks per month or less at night), in persistentform (permanent attacks during daytime, frequent attacks at night) andin any intermediate form. Within the meaning of the application the termasthma relates to non-allergic as well as to allergic asthma. Preferredembodiments of the invention relate to allergic asthma, includingIgE-mediated and non IgE-mediated forms of asthma. Specificallypreferred embodiments relate to IgE-mediated allergic asthma, mostpreferably to atopic asthma.

“atopic asthma”: IgE-mediated form of asthma in patients with a geneticpredisposition which often occurs in conjunction with atopic eczema andIgE-mediated allergies, for example pollen allergy (hay fever), housedust or dust mite.

“dermatitis”: The term “dermatitis” relates to local inflammation of theskin and encompasses, besides other forms, “eczema” and “contactdermatitis” (see definitions below). Preferred embodiments of theinvention relate to dermatitis, preferably to eczema and contactdermatitis.

“eczema”: The term “eczema” relates to the atopic eczema/dermatitissyndrome (AEDS), describing an aggregation of several skin diseases withcertain clinical characteristics in common involving a geneticallydetermined skin barrier et defect. This genetically determined targetorgan sensitivity constitutes the basis for eczema. In children andyoung adults of the atopic constitution, the underlying inflammation isdominated by an IgE-antibody associated reaction (atopic eczema). Inchronic cases, the inflammation seems to be less influenced by IgEantibody, and the dominating cells in biopsies are lymphocytes. Eczemarelates to non-allergic eczema and allergic eczema. Preferredembodiments of the invention relate to eczema, preferably allergiceczema including atopic (IgE-mediated) eczema and non atopic forms ofeczema. Most preferably, the invention relates to atopic (IgE-mediated)eczema.

“contact dermatitis”: The term “contact dermatitis” relates to localinflammatory reaction in the skin caused by close contact with lowmolecular weight chemicals or irritants. Contact dermatitis can be ofallergic as well as non-allergic nature. Allergic contact dermatitis ismediated by immunological mechanisms, predominantly TH1 lymphocytes.Typical allergens acting as haptens and causing allergic contactdermatitis are nickel, chromium ions, fragrances, preservatives, andurushiol, from the poison ivy plant. Exposure can occur through oraluptake, so-called systemic allergic contact dermatitis. A subgroup ofcontact dermatitis, protein contact dermatitis, is an IgE-associatedreaction caused by absorption of proteins through damaged skin.Preferred embodiments of the invention relate to contact dermatitis,preferably allergic contact dermatitis. Further preferred embodimentsrelate to protein contact dermatitis.

“urticaria”: The term “urticaria” relates to a non inflammatory reactionin the skin caused by an irritant or allergen and includes non-allergicurticaria as well as allergic urticaria. Allergic urticaria is mediatedby immunological mechanisms, which commonly is IgE-mediated but can alsobe immune complex-associated. Urticaria can also develop locally aftertopical contact with the allergen, as occurs on the hands of a personwith latex allergy wearing latex gloves or in a person with dog allergylicked by a dog. Preferred embodiments of the invention relate tourticaria, preferably allergic urticaria, most preferably IgE-mediatedallergic urticaria.

“food hypersensitivity”: The term “food hypersensitivity” relates toadverse reaction to food, which can be of non-allergic as well asallergic nature. Allergic food hypersensitivities can be IgE-madiatedand are referred to as food allergies. Severe, generalized allergicreactions to food can be classified as anaphylaxis (see below).Preferred embodiments of the invention relate to food allergy,preferably to IgE-mediated food allergy.

“drug hypersensitivity”: The term “drug hypersensitivity” relates tohypersensitive reactions of the body towards drugs which can be ofnon-allergic as well as of allergic nature. When immunologic mechanismshave been shown, either antibody or cell mediated, the reactions arereferred to as drug allergy. Drug allergies can be mediated by IgE.Preferred embodiments of the invention relate to drug hypersensitivity,preferably to drug allergy, most preferably to IgE-mediated drugallergy.

“Insect sting hypersensitivity” or “Insect bite hypersensitivity”: theseterms relate to hypersensitive reactions towards insect venom and salivawhich can be of non-allergic as well as allergic nature. Insect stinghypersensitivity or insect bite hypersensitivity mediated by animmunologic mechanism is referred to as venom or saliva allergy, as inbee venom allergy. The large quantity of venom allergen in a sting iscomparable with years of inhaled pollen allergen. This high-dosesensitization probably explains why there is no need for a geneticpredisposition for developing such an allergy. Preferred embodiments ofthe invention relate to venom allergy, preferably to IgE-mediated venomallergy, most preferably to IgE mediated bee venom allergy.

“anaphylaxis”: The term “anaphylaxis” refers to a severe,life-threatening, generalized or systemic hypersensitive reaction. Thereaction usually develops gradually, most often starting with itching ofthe gums/throat, the palms, or the soles, and local urticaria;developing to a multiple organ reaction often dominated by severeasthma; and culminating in hypotension and shock. Hypotension and severebronchospasm do not have to be present for a reaction to be classifiedas anaphylaxis. Anaphylaxis can be of non-allergic as well as ofallergic nature. Allergic anaphylaxis involves an immunologic mechanismlike an IgG immune complex, complement related, or immune cell-mediatedmechanism. Anaphylaxis preferably relates to an anaphylactic reactionmediated by IgE antibodies (IgE-mediated anaphylaxis), most preferablyto peanut-induced food anaphylaxis or bee venom-induced anaphylaxis.

“allergen”: The term “allergen” refers to a substance causing allergy.Preferred allergens are allergens disclosed in Shough, H. et al.,REMINGTON'S PHARMACEUTICAL SCIENCES, 19th edition, (Chap. 82), MackPublishing Company, Mack Publishing Group, Easton, Pa. (1995), theentire contents of which is hereby incorporated by reference. Allergensserve as antigens in vertebrate animals. The term “allergen”, as usedherein, also refers to “allergen extracts” and “allergenic epitopes.”Very preferred allergens are selected from the group consisting of:pollens (e.g. grass, ragweed, birch and mountain cedar); house dust anddust mites; mammalian epidermal allergens and animal danders; mold andfungus: insect bodies and insect venom; feathers; food; and drugs (e.g.penicillin).

“allergen extracts”/“provocation test solutions”: Allergen extracts arecomponents of provocation test solutions to be used for conjunctival,nasal and bronchial challenges. Such allergen extracts are commerciallyavailable and methods for producing such extracts are well-known.Preferred are single allergen provocation solutions comprising a singleallergen extract which is prepared from a source selected from the groupconsisting of (i) tree species or a grass species, most preferablyselected from the group consisting of alder, ash, birch, hazel, orchardgrass, velvet grass, rye grass, timothy grass, Kentucky blue grass,Meadow fescue, Bermuda grass, ragweed, rye and wheat; (ii) epithelia ofdifferent animal species, preferably epithelia from an animal speciesselected from the group consisting of cat, dog and horse; (iii) moulds,preferably moulds selected from the group consisting of aspergillus,candida, alternaria, and saccharomyces; and (iv) mite species,preferably mite species selected from the group consisting ofDermatophagoides farinae, Dermatophagoides pteronyssinus and Acarussiro. Allergen extracts comprising allergen mixtures can also be used inprovocation test solutions. Preferred are allergen mixtures of differentgrasses, preferably of orchard grass, velvet grass, rye grass, timothygrass, Kentucky blue grass and/or Meadow fescue. Further preferred areallergen mixtures of grasses, cereals, different trees and/or animalhair. Provocation solutions are usually prepared in physiological salineand can be preserved by addition of 0.4% phenol.

“antibody”: As used herein, the term “antibody” refers to moleculesbelonging to the class of immunoglobulins which are capable of bindingan epitope or antigenic determinant.

“antigen”: As used herein, the term “antigen” refers to a moleculecapable of being bound by an antibody or a T cell receptor (TCR) ifpresented by MHC molecules. The term “antigen”, as used herein, alsoencompasses T-cell epitopes. An antigen is additionally capable of beingrecognized by the immune system and/or being capable of inducing ahumoral immune response and/or cellular immune response leading to theactivation of B- and/or T-lymphocytes. Antigens as used herein may alsobe mixtures of several individual antigens. However “antigen” does notencompass any of the components of the compositions of the invention. Inparticular, the term “antigen” does not refer to the particle nor to theISS-NA of the invention. The term “antigen” also does not refer to anycomponent forming the particle of the invention, such as, for examplecapsid protein.

“epitope”: As used herein, the term “epitope” refers to continuous ordiscontinuous portions of a polypeptide having antigenic or immunogenicactivity in an animal, preferably a mammal, and most preferably in ahuman. An epitope is recognized by an antibody or a T cell through its Tcell receptor in the context of an MHC molecule.

“Immune response”: As used herein, the term “immune response” refers toa humoral immune response and/or cellular immune response leading to theactivation or proliferation of B- and/or T-lymphocytes and/or antigenpresenting cells and I or other cells of the innate immune system, suchas pDC. Alternatively, the immune response may also result in an alteredfunction of effector cells, such as mast cells.

“Inducing an immune response”: A substance, preferably an ISS-NA iscapable of inducing an immune response when upon exposure of a cell oran organism to said substance, preferably to an effective amount of saidsubstance, an immune response is detectable in said cell or animal whichdoes not occur in the untreated control.

“stimulating production of IFN-alpha”: The production of IFN-alpha by acell, preferably by a dendritic cell, after exposure to a specificsubstance is a strong indication of an immunostimulatory effect of saidsubstance. Therefore, ISS-NA which are capable of inducing theproduction of IFN-alpha are preferred in the context of the invention.IFN-alpha production by a cell can be determined by various methodsgenerally known in the art, preferably by a method selected from (a)ELISA, most preferably by ELISA essentially as described in Example 14;(b) flow cytometry analysis using fluorochrom-conjugated antibodies,preferably as described in Example 14; and (c) cytopathicity inhibitionbioassays. A typical cytopathicity inhibition bioassay is based onbovine MDBK cells infected with vesicular stomatitis virus, aspreviously described in Pestka, S. (1986) “Interferon Standards andGeneral Abbreviations”, in Methods in Enzymology, Academic Press, NewYork 119, 14-23. In the context of the application a substance,preferably an immunostimulatory nucleic acid, is regarded as being“capable of stimulating IFN-alpha production”, when the production ofIFN-alpha by a cell as detected by any one of the above describedmethods, preferably by ELISA, most preferably as described in Example14, is significantly increased upon exposure of said cell to saidsubstance as compared to a control cell, wherein typically andpreferably, said IFN-alpha production is increased by a factor of atleast about 2, more preferably by a factor of about 3 or more.

“enhancing an Immune response”: A substance which enhances an immuneresponse, refers to a substance, preferably to an ISS-NA, which iscapable of intensifying or modulating the immune response of a cell oran animal upon exposure of said cell or said animal to said substance,as compared to a suitable control. This observation can relate to anyparameter known in the art to be indicative for an immune response,preferably to the formation of cytokines and to cytotoxicity. Forexample, the lytic activity of cytotoxic T cells can be measured, e.g.using a ⁵¹Cr release assay, with and without the substance, preferablythe ISS-NA. The amount of the substance at which the CTL lytic activityis enhanced as compared to the CTL lytic activity without the substanceis said to be an amount sufficient to enhance the immune response. In apreferred embodiment, the immune response is enhanced by a factor of atleast about 2, more preferably by a factor of about 3 or more. Theamount or type of cytokines secreted may also be altered. Alternatively,the amount of antibodies induced or their subclasses may be altered.

“Immunostimulatory nucleic acid (ISS-NA)”: As used herein, the termimmunostimulatory nucleic acid refers to a nucleic acid capable ofinducing and/or enhancing an immune response. ISS-NA, as used herein,comprise ribonucleic acids and in particular desoxyribonucleic acids,wherein both, ribonucleic acids and desoxyribonucleic acids may beeither double stranded or single stranded. Preferred ISS-NA aredesoxyribonucleic acids, wherein further preferably saiddesoxyribonucleic acids are single stranded. Preferably, ISS-NA containat least one CpG motif comprising an unmethylated C. Very preferredISS-NA comprise at least one CpG motif, wherein said at least one CpGmotif comprises or preferably consist of at least one, preferably one,CG dinucleotide, wherein the C is unmethylated. Preferably, but notnecessarily, said CG dinucleotide is part of a palindromic sequence.ISS-NA not containing CpG motifs as described above encompass, by way ofexample, nucleic acids lacking CG dinucleotides, as well as nucleicacids containing CG dinucleotides with a methylated C. The term“immunostimulatory nucleic acid” as used herein also refers to nucleicacids that contain modified bases, preferably 4-bromo-cytosine.Specifically preferred in the context of the invention are ISS-NA whichare capable of stimulating IFN-alpha production in dendritic cells.

“oligonucleotide”: As used herein, the term “oligonucleotide” refers toa nucleic acid sequence comprising 2 or more nucleotides, preferably atleast about 6 nucleotides to about 100,000 nucleotides, more preferablyabout 6 to about 2000 nucleotides, and still more preferably about 6 toabout 300 nucleotides, even more preferably about 20 to about 300nucleotides, and even more preferably about 20 to about 100 nucleotides,and most preferably 20 to 40 nucleotides. Very preferablyoligonucleotides comprise about 30 nucleotides, more preferablyoligonucleotides comprise exactly 30 nucleotides, and most preferablyoligonucleotides consist of exactly 30 nucleotides. The termoligonucleotide also refers to a nucleic acid comprising more than 100to about 2000 nucleotides, preferably more than 100 to about 1000nucleotides, and more preferably more than 100 to about 500 nucleotides.

Oligonucleotides are polyribonucleotides or polydeoxyribonucleotides andare preferably selected from (a) unmodified RNA or DNA, and (b) modifiedRNA or DNA. The modification may comprise the backbone or nucleotideanalogues. Oligonucleotides are preferably selected from the groupconsisting of (a) single- and double-stranded DNA, (b) DNA that is amixture of single- and double-stranded regions, (c) single- anddouble-stranded RNA, (d) RNA that is mixture of single- anddouble-stranded regions, and (e) hybrid molecules comprising DNA and RNAthat are single-stranded or, more preferably, double-stranded or amixture of single- and double-stranded regions. In a further embodimentoligonucleotides are triple-stranded regions and higher-orderedstructures comprising RNA or DNA or both RNA and DNA. In furtherembodiments oligonucleotide are synthetic, genomic or recombinant.Preferred oligonucleotides are selected from the group consisting ofλ-DNA, cosmid DNA, artificial bacterial chromosome, yeast artificialchromosome and filamentous bacteriophage, preferably M13. In oneembodiment oligonucleotide refers to (a) DNA or RNA containing at leastone modified nucleotide or at least one nucleotide analogue, or (b) toDNA or RNA with backbones modified for stability or for other reasons.Preferred nucleotide modifications/analogs are selected from the groupconsisting of (a) peptide nucleic acid, (b) inosin, (c) tritylatedbases, (d) phosphorothioates, (e) alkylphosphorothioates. (f)5-nitroindole desoxyribofuranosyl, (g) 5-methyldesoxycytosine, and (h)5,6-dihydro-5,6-dihydroxydesoxythymidine. Phosphothioated nucleotidesare protected against degradation in a cell or an organism and aretherefore preferred nucleotide modifications. Further preferred arechemically, enzymatically or metabolically modified forms ofpolynucleotides as typically found in nature, as well as the chemicalforms of DNA and RNA characteristic of viruses and cells. Othernucleotide analogs or modifications will be evident to those skilled inthe art. However, unmodified oligonucleotides consisting exclusively ofphosphodiester bound nucleotides, typically are more active as ISS-NAthan modified nucleotides and are therefore generally preferred in thecontext of the invention. Most preferred are oligonucleotides consistingexclusively of phosphodiester bound deoxinucleotides. Further preferredare oligonucleotides capable of stimulating IFN-alpha production incells, preferably in dendritic cells. Very preferred oligonucleotidescapable of stimulating IFN-alpha production in cells are selected fromA-type CpGs and C-type CpGs.

“CpG motif”: As used herein, the term “CpG motif” refers to a pattern ofnucleotides that includes an unmethylated central CpG, i.e. theunmethylated CpG dinucleotide, in which the C is unmethylated,surrounded by at least one base, preferably one or two nucleotides,flanking (on the 3′ and the 5′ side of) the central CpG. Typically andpreferably, the CpG motif as used herein, comprises or alternativelyconsists of the unmethylated CpG dinucleotide and two nucleotides on its5′ and 3′ ends. Without being bound by theory, the bases flanking theCpG confer a significant part of the activity to the CpGoligonucleotide.

“CpG”/“unmethylated CpG-containing oligonucleotide”: As used herein, theterm “unmethylated CpG-containing oligonucleotide” or “CpG” refers to anoligonucleoctide, preferably to an oligodesoxynucleotide, containing atleast one CpG motif. Thus, a CpG contains at least one unmethylatedcytosine, guanine dinucleotide. Preferred CpGs stimulate/activate, e.g.have a mitogenic effect on, or induce or increase cytokine expressionby, a vertebrate bone marrow derived cell. For example, CpGs can beuseful in activating B cells, NK cells and antigen-presenting cells,such as dendritic cells, monocytes and macrophages. Preferably, CpGrelates to an oligodesoxynucleotide, preferably to a single strandedoligodesoxynucleotide, containing an unmethylated cytosine followed 3′by a guanosine, wherein said unmethylated cytosine and said guanosineare linked by a phosphate bond, wherein preferably said phosphate boundis a phosphodiester bound or a phosphothioate bound, and wherein furtherpreferably said phosphate bond is a phosphodiester bound. CpGs caninclude nucleotide analogs such as analogs containing phosphorothioesterbonds and can be double-stranded or single-stranded. Generally,double-stranded molecules are more stable in vivo, while single-strandedmolecules have increased immune activity. Preferably, as used herein, aCpG is an oligonucleotide that is at least about ten nucleotides inlength and comprises at least one CpG motif, wherein further preferablysaid CpG is 10 to 60, more preferably 15 to 50, still more preferably 20to 40, still more preferably about 30, and most preferably exactly 30nucleotides in length. A CpG may consist of methylated and/orunmethylated nucleotides, wherein said at least one CpG motif comprisesat least one CG dinucleotide wherein the C is unmethylated. The CpG mayalso comprise methylated and unmethylated sequence stretches, whereinsaid at least one CpG motif comprises at least one CG dinucleotidewherein the C is unmethylated. Very preferred CpGs consist exclusivelyof unmethylated nucleotides. Very preferably, CpG relates to a singlestranded oligodesoxynucleotide containing an unmethylated cytosinefollowed 3′ by a guanosine, wherein said unmethylated cytosine and saidguanosine are linked by a phosphodiester bound. Still more preferably,CpG relates to a single stranded oligodesoxynucleotide containing anunmethylated cytosine followed 3′ by a guanosine, wherein saidunmethylated cytosine and said guanosine are linked by a phosphodiesterbound, and wherein said CpG consist exclusively of unmethylatednucleotides. Most preferably, CpG relates to a single strandedoligodesoxynucleotide of about 30 nucleotides in length, containing anunmethylated cytosine followed 3′ by a guanosine, wherein saidunmethylated cytosine and said guanosine are linked by a phosphodiesterbound, and wherein said CpG consist exclusively of unmethylatednucleotides. The CpGs can include nucleotide analogs such as analogscontaining phosphorothioester bonds and can be double-stranded orsingle-stranded. Generally, phosphodiester CpGs are A-type CpGs asindicated below, while phosphothioester stabilized CpGs are B-type CpGsor C-type CpGs. Preferred CpG oligonucleotides in the context of theinvention are A-type CpGs and C-type CpG, most preferred are A-typeCpGs.

“A-type CpG”: As used herein, the term “A-type CpG” or “D-type CpG”refers to an oligodesoxynucleotide (ODN) comprising at least one CpGmotif. A-type CpGs preferentially stimulate activation of T cells andthe maturation of dendritic cells and are capable of stimulatingIFN-alpha production. In A-type CpGs, the nucleotides of the at leastone CpG motif are linked by at least one phosphodiester bond. A-typeCpGs comprise at least one phosphodiester bond CpG motif which may beflanked at its 5′ end and/or, preferably and, at its 3′ end byphosphorothioate bound nucleotides. Preferably, the CpG motif, andhereby preferably the CG dinucleotide and its immediate flanking regionscomprising at least one, preferably two nucleotides, are composed ofphosphodiester nucleotides. Preferred A-type CpGs exclusively consist ofphosphodiester (PO) bond nucleotides. Further preferred A-type CpGs donot comprise phosphothioate bounds. Typically and preferably, the term“A-type CpG” or “D-type CpG” as used within this specification, refersto an oligodesoxynucleotide (ODN) comprising at least one CpG motif andhaving poly G motifs at the 5′ and/or 3′ ends. Typically and preferably,the poly G motif comprises or alternatively consists of at least one,preferably at least three, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 Gs (guanosines), most preferably by at least 10 Gs. In someembodiments, the 5′ and/or 3′ ends, typically and preferably at leastone G of the poly G motifs at the 5′ and/or 3′ ends, preferably at leasttwo, three or four, even more preferably all Gs of the poly G motif, arephoshorothioate modified. However, in a very preferred embodiment, allGs of the poly G motif are linked by phosphodiester bonds. Preferably,the A-type CpG of the invention comprises or alternatively consists of apalindromic sequence. Typically and preferably, the CpG motif is part ofsaid palindromic sequence. Typically and preferably, all nucleotides,but at least the CpG motif of the palindromic sequence, are composed ofphosphodiester nucleotides. Typically and preferably, the palindromicsequence is SEQ ID NO:28. Very preferred A-type CpGs are 16 to 30nucleotides in length, consist exclusively of phosphodiester boundnucleotides, comprise a palindromic sequence, preferably the palindromicsequence of SEQ ID NO:28, and are flanked at their 5′ and at their 3′end by a poly G motif consisting of 3 to 10 Gs.

“B-type CpG”: As used herein, the term “B-type CpG” (K-type) relates toa CpG oligonucleotide which predominantly or preferably exclusivelyconsists of modified nucleotides, preferably phosphorothioate modifiednucleotides. B-type CpGs stimulate preferentially B-cell and to someextent NK-cell activation and cytokine production.

“C-type CpG”: As used herein, the term “C-type CpG” relates to a CpGoligonucleotide which like a B-type oligonucleotide predominantly orpreferably exclusively consists of modified nucleotides, preferablyphosphorothioate modified nucleotides. Examples of C-type CpGs have beendescribed in WO2005/042018A2 and in Vollmer et al. 2004, Eur. J.Immunol. 43:351-262 which are incorporated herein by reference. Specificreference is made to SEQ IDs NO: 1 to 69 of WO2005/042018A2. C-type CpGscombine effects of A-type and B-type CpGs and stimulate B-cell orNK-cell activation and IFN-alpha production, preferably IFN-alphaproduction in dendritic cells. C-type CpCs which are capable ofstimulating IFN-alpha production, preferably in dendritic cells, aregenerally preferred in the context of the invention. In contrast toA-type CpGs, C-type CpGs do not typically comprise poly-G stretches.C-type CpGs preferably comprise or alternatively consist of palindromicsequences comprising CpG motifs, preferably palindromic sequences asdepicted in SEQ ID NOs:53 to 60. Further preferred C-type CpGs comprisea sequence selected from the group consisting of (a) TCGTCGITTA (SEQ IDNO:61), (b) CGGCGCCGTGCCG (SEQ ID NO:62) and (c) CGGCGTCGTGCCG (SEQ IDNO:63), wherein the 5′ end of said C-type CpG preferably consists of SEQID NO:61 and/or wherein the 3′ end of said C-type CpG preferablyconsists of a nucleotide sequence selected from SEQ ID NO:62 and SEQ IDNO:63, most preferably the 3′ end of said C-type CpG consists of SEQ IDNO:63. Further preferred C-type CpGs are selected from the groupconsisting of (a) TCGTCGTTTTACGGCGCCGTGCCG (SEQ ID NO:64) and (b)TCGTCGTTTTACGGCGTCGTGCCG (SEQ ID NO:65), wherein preferably all nucleicacids of said C-type CpGs are phosphorothioate bound. Further preferredC-type CpGs are selected from the group consisting of (a)TCpGTCGTTTTACGGCGCCGTGCCG (SEQ ID NO:64); (b) TCGTCGTTTACpGGCpGCCpGTGCCG(SEQ ID NO:64); (c) TCGTCGTTT TACpGGCGCCpGTGCCG (SEQ ID NO:64); (d)TCGTCpGTITTACpGGCGCCpGTGCCG (SEQ ID NO:64); wherein p indicatesphosphodiester bounds while all other nucleotides are phosphorothioatebound. C-type CpGs selected from the group consisting of (a)TCGTCGTTTTCOGCGCGCGCCG (SEQ ID NO:66); (b) TCGTCGTTTTCGACGGCCGTCG (SEQID NO:67); (c) TCGTCGTTTTCCGGCGCGCCGG (SEQ ID NO:68); (d)TCGTCGTTTTCGGCGCGCGTCG (SEQ ID NO:69); (e) TCGGCGCGCGCCGTCGTCGTTT (SEQID NO:70); (f) TTGGCGCGCGCCGTCGTCGTTT (SEQ ID NO:71); (g)TCGTCGTTTTCGTCGGCCGCCG (SEQ ID NO:72); (h) TCGTCGTTTTCGGCTTTTGCCG (SEQID NO:73); (i) TCGTCGTTTTCGGCGTTTTTTT (SEQ ID NO:74); and (j)TCGTCGTTTTCGGCGGCCGCCG (SEQ ID NO:75) are potent inducers of IFN-alphaproduction (Vollmer et al. 2004, Eur. J. Immunol. 43:351-262, p. 253,see Table 1 therein) and are thus specifically preferredimmunostimulatory nucleic acids in the context of the invention.

“palindromic sequence”: A palindromic sequences is a nucleotide sequencewhich, when existing in the form of a double stranded nucleic acid withregular base pairing (A/T; C/G), would consist of two single strandswith identical sequence in 5′-3′ direction. An immunostimulatory nucleicacids of the invention preferably comprises a palindromic sequence,preferably a palindromic sequence consisting of at least 6, preferablyof at least 7, 8, 9 or 10, most preferably of exactly 10 nucleotides,wherein most preferably said palindromic sequence preferably comprises aCpG motif. Palindromic sequences of immunostimulatory nucleic acidsuseful in the context of the invention are, for example, described inYamamoto et al. 1992, J. Immunol. 148(12):4072-4076 and Kuramoto et al.1992, Jpn. J. Cancer Res. 83:1128-1131. Preferred palindromic sequencescomprise a CpG motif and are selected from the group consisting of (a)GACGTC (SEQ ID NO:35), (b) AGCGCT (SEQ ID NO:36), (c) AACGTT (SEQ IDNO:37), (d) ATCGAT (SEQ ID NO:38); (e) CGATCG (SEQ ID NO:39); (f) CGTACG(SEQ ID NO:40); (g) CGCGCG (SEQ ID NO:41); (h) GCGCGC (SEQ ID NO:42);(i) TCGCGA (SEQ ID NO:43); (j) ACGATCGT (SEQ ID NO:44); (k) CGACGATCGTCG(SEQ ID NO:45); (l) CGACGACGATCGTCGTCG (SEQ ID NO:46); (m) GACGATCGTC(SEQ ID NO:28), (n) CGACGACGATCGTCGTCG (SEQ ID NO:47); (O) AACGTT (SEQID NO:48); (p) CAACGTTG (SEQ ID NO:49); (q) ACAACOTTGT (SEQ ID NO:50);(r) AACAACGTTGTT (SEQ ID NO:51); (s) CAACAACGTTGTTG (SEQ ID NO:52); (t)CGGCGCGCGCCG (SEQ ID NO:53); (u) CGACGGCCGTCG (SEQ ID NO:54); (v)CCGGCGCOCCGG (SEQ ID NO:55); (w) CGCGCG (SEQ ID NO:56), (x) CGGCGCGCGCCG(SEQ ID NO:57); (y) GGCGCGCGCC (SEQ ID NO:58); (z) CGGCCG (SEQ IDNO:59); and (aa) CGGCGGCCGCCG (SEQ ID NO:60). A-type CpGs preferablycomprise a palindromic sequence selected from palindromic sequences (a)to (s), while C-type CpGs preferably comprise a palindromic sequenceselected from palindromic sequences (t) to (aa).

“packaged”: The term “packaged” as used herein refers to the state of anISS-NA, in particular an unmethylated CpG-containing oligonucleotide, inrelation to the particle, in particular the VLP. The term “packaged” asused herein refers to covalent binding, preferably by chemicallycoupling. More preferably, the term “packaged” refers to non-covalentbinding, preferably to ionic interactions, hydrophobic interactions, orhydrogen bonds. Covalent bonds are preferably selected from the groupconsisting of ester, ether, phosphoester, amide, peptide, imide,carbon-sulfur bonds, and carbon-phosphorus bonds. Very preferably, theterm “packaged” as used herein refers to the enclosement, or partialenclosement, of said ISS-NA within the particle. For example, theunmethylated CpG-containing oligonucleotide can be enclosed by the VLPwithout the existence of an actual binding, neither covalently nornon-covalently, or with a non-covalent binding.

Typically and preferably, a particle packaged with ISS-NA protects saidISS-NA from degradation, preferably from DNAse or RNAse hydrolysis.Therefore, in the preferred meaning, the term “packaged” indicates thatthe ISS-NA, preferably the unmethylated CpG-containing oligonucleotide,in a packaged state is not accessible to DNAse or RNAse hydrolysis. Morepreferably, the term “packaged” indicates that the ISS-NA, preferablythe unmethylated CpG-containing oligonucleotide, is not accessible toDNAse hydrolysis, wherein further preferably the DNAse is DNAseI orBenzonase. Still more preferably, the term “packaged” indicates that theunmethylated CpG-containing oligonucleotide is not accessible toBenzonase hydrolysis.

The accessibility of the ISS-NA, in particular the of the unmethyatedCpG-containing oligonucleotide for DNAse (e.g. DNaseI or Benzonase) ispreferably assayed as described in Examples 11-17 of WO2003/024481A2(see p. 111 therein). In a preferred meaning, a VLP is regarded as beingpackaged with an unmethylated CpG-containing oligonucleotide, when aftertreatment with Benzonase (190 U Benzonase 1 mg capsid protein in abuffer comprising 2 mM MgCl₂, pH 7.2, 20-25° C., 18 h) at least 90%,preferably at least 95%, most preferably at least 98% of saidunmethylated CpG-containing oligonucleotide can be recovered from saidVLP (e.g. in an ethidiumbromide stained gel). It is apparent for theartisan that such assays require appropriate controls and may need to beadapted to the specific combination of VLP and unmethylatedCpG-containing oligonucleotide. In a more preferred meaning, a VLP of anRNA bacteriophage is regarded as being packaged with an unmethylatedCpG-containing oligonucleotide, when after treatment with Benzonase (190U Benzonase/mg capsid protein in a buffer comprising 2 mM MgCl₂, pH 7.2,20-25° C., 18 h) at least 90%, preferably at least 95%, most preferablyat least 98% of said unmethylated CpG-containing oligonucleotide can berecovered from said VLP of an RNA bacteriophage. In a very preferredmeaning, a VLP of a RNA bacteriophage is regarded as being packaged withG10 (SEQ ID NO:27) oligonucleotide, when after treatment with Benzonase(190 U Benzonase/mg capsid protein in a buffer comprising 2 mM MgCl₂, pH7.2, 20-25° C., 18 h) at least 90%, preferably at least 95%, mostpreferably at least 98% of said unmethylated CpG-containingoligonucleotide can be recovered from said VLP of an RNA bacteriophage.In more specific meaning, a VLP of a RNA bacteriophage Qβ, AP205, GA orfr is regarded as being packaged with G10 (SEQ ID NO:27)oligonucleotide, when after treatment with Benzonase (190 U Benzonase/mgcapsid protein in a buffer comprising 2 mM MgCl₂, pH 7.2, 20-25° C., 18h) at least 90%, preferably at least 95%, most preferably at least 98%of said unmethylated CpG-containing oligonucleotide can be recoveredfrom said VLP of an RNA bacteriophage. In a very specific meaning, a VLPof a RNA bacteriophage Qβ is regarded as being packaged with G10 (SEQ IDNO:27) oligonucleotide, when after treatment with Benzonase (190 UBenzonase/mg capsid protein in a buffer comprising 2 mM MgCl₂, pH 7.2,20-25° C., 18 h) at least 90%, preferably at least 95%, most preferablyat least 98% of said unmethylated CpG-containing oligonucleotide can berecovered from said VLP of RNA bacteriophage Qβ.

Alternatively, the packaging state of an ISS-NA in a particle, inparticular of ISS-NA which do not constitute a substrate for DNAse orRNAse hydrolysis, can be assessed by size exclusion chromatography orSDS-PAGE and subsequent spectroscopic analysis as described in Example4. A further possibility to verify the packaging state of a particlepackaged with an ISS-NA is dialysis or tangential flow filtration of theparticle, for example under conditions as described in Example 1,wherein non-packaged nucleic acids are removed while packaged nucleicaids remain associated with said particle.

In the very preferred meaning, and wherein the particle is a virusparticle or a virus-like particle of a bacteriophage, preferably of anRNA-bacteriophage, further preferably of an RNA bacteriophage Qβ, andmost preferably of a virus-like particle of a RNA-bacteriophage Qβ, andwherein said ISS-NA is a unmethylated CpG-containing oligonucleotide,preferably a A-type CpG, further preferably the SEQ ID NO:27, the term“packaged” indicates that the particle packaged with said ISS-NA elutesat the same retention time as the virus-like particle of saidbacteriophage, preferably of said RNA-bacteriophage, further preferablyof said RNA bacteriophage Qβ obtained by recombinant expression of thecoat protein in E. coli, preferably wherein said retention time isdetermined by size exclusion chromatography, preferably as described inExample 4 of the present application, and comprises said ISS-NA asdetermined preferably as described in Example 4 of the presentapplication.

In preferred embodiments, the ISS-NA, preferably the unmethylatedCpG-containing oligonucleotide, is packaged inside the particle,preferably VLP capsids, most preferably in a non-covalent manner.Protocols for the preparation of VLPs packaged with unmethylatedCpG-containing oligonucleotide are provided in the prior art, e.g. inWO2003/024481 A2 (see Examples 2, 3, 7, 8, 10, 11, 12, 13, 14, 15, 16,and 17 therein, in particular Examples 14-17 therein) andWO2004/000351A1. The disclosure of both publications is incorporated tothis application by reference. Further Protocols for the preparation ofVLPs packaged with unmethylated CpG-containing oligonucleotide areprovided Examples 1, 3 5 and 6 of the present application.

It is to be understood that under the assay conditions specified above,especially those of Examples 11-17 of WO2003/024481A2, some syntheticparticles which are packaged with said ISS-NA may release a certainlimited amount of ISS-NA, wherein said release typically follows a bi-or more phasic kinetic, wherein said kinetic may comprise a fast initialburst release phase and at least one slow release phase. For example, acertain percentage of the ISS-NA packaged in a synthetic particle may bereleased in a burst release phase, when incubated at 37° C. or 30° C. inphysiological buffers, in vitro. In this case the burst release phase isfollowed by at least one slow release phase. In some cases the secondrelease phase will be flat, meaning that no or very limited amounts ofISS-NA are released from the synthetic particle in that phase. The twoor more phases are identified by examination of the release kinetic ofISS-NA from the synthetic particle. Typically, an initial burst releasephase will be complete in a few hours but may last up to 24 hours, whilethe slow release phase may last from 2-3 days up to 6 days or longer. Insome cases, the slow release phase may be nearly flat, with no or verylittle ISS-NA released after the burst release phase. Alternatively,there may be no initial burst phase, but rather one or more slow releasephases. As the burst release phase may be concomitant to nuclease orserum exposure in an assay to assess packaging, protection fromdegradation by nucleases or serum assessed in this assay may not becomplete. In the instance of an initial burst release phase ofoligonucleotide under the conditions used in the assays to testprotection, protection is assessed during the time span of the assaycorresponding to the slow release phase. Thus, the term “packaged” asrelated in particles being synthetic particles but not being virusparticles or virus-like particles also encompasses compositionscomprising, essentially consisting of, or consisting of syntheticparticles and ISS-NA's, in which such release of ISS-NA by saidsynthetic particle takes place, provided that at least 30%, preferablyat least 40%, more preferably at least 50%, still more preferably atleast 60%, even more preferably at least 70%, and most preferably atleast 80% of the ISS-NA packaged in said synthetic particle is remainingassociated with said synthetic particle at the end of the assay.

“particle”: Particles of the invention have a diameter of 10 to 10000nm, preferably of 20 to 1000 nm, more preferably 20 to 500 nm, stillmore preferably 20 to 300 nm, still more preferably 20 to 200 nm, andmost preferably 20 to 100 nm, wherein these particles can preferably bepackaged with an ISS-NA. Particles of the invention are preferablyselected from the group consisting of synthetic particles and VLPs. Verypreferred particles of the invention are VLPs, most preferably VLPs ofRNA phages.

“size of a particle”: The size of a spherical or nearly sphericalparticle is determined as the medium diameter of a population ofparticles; the size of an elliptic, longitudinal or irregular formedparticle refers to the arithmetic medium of the longest axis in apopulation of particles. Typically and preferably, the size of aparticle, preferably of a nanoparticle or a microparticle, mostpreferably of a nanoparticle, is determined dynamic light scattering(DLS) technology (Example 13).

“synthetic particles”: As used herein, “synthetic particle” refers toparticles which are formed by chemical or physical processes, preferablyby polymerization of monomers, precipitation of polymers, assembly ofmacromolecules brought together, for example by aggregation or heatdenaturation, chemical cross-linking of said assembled macromolecules.Preferred synthetic particles are selected from liposomes,microparticles, and nanoparticles. Further preferred synthetic particlesare selected from liposomes, microparticles, nanoparticles, andvirosomes. Very preferred synthetic particles are nanoparticles. Theterm “synthetic particles” does not refer to particles formed byassembly of viral proteins. Particles formed from viral coat proteinsare specifically referred to as virus particles or virus-like particles.Viroids, retrotransposon particles, and all particles formed fromgenetically encoded viral proteins are also understood as virusparticles or virus-like particles.

“liposomes”: As used herein, the term “liposome” refers to phospholipidvesicles comprising one or more, preferably one, two, or threephospholipid bilayer membranes. Liposomes vary in charge and in sizedepending on the method of preparation and the lipids used. The liposomeof the present invention may be neutral, cationic, anionic, stealth, orcationic stealth. Preferably, the liposome of the invention is acationic liposome. The liposome may have a diameter between 100 and 800nm, preferably between 100 and 400 nm, more preferably between 100 and300 nm, even more preferably between 100 and 200 nm, most preferablyless than 200 nm. The term “liposome”, as used herein, shall alsoencompass modified liposomes, preferably modified liposomes, wherein thesurface of the liposomes may be specifically modified to optimizebinding to DC, for example, via specific sugar moieties (Fukasawa etal., (1998), FEBS, 441, 353-356) or antibodies (Serre et al. (1998), J.Immunol., 161, 6059-6067).

“lipopolyplex”: Lipopolyplex particles are liposomes comprising or,preferably, essentially consisting of, most preferably consisting of acationic lipid, a polycation and ISS-NA or DNA, whereby it is thoughtthat the cationic lipid forms an additional protective layer surroundingthe complex of ISS-NA or DNA with the polycation (Pelisek J. et al. JGene Med 2006; 8:186-197).

“microparticle”: As used herein, “microparticle” refers to syntheticparticles of controlled dimension in the order of micrometers (i.e. >1μm, and <1000 μm). Preferred microparticles have a size of 1 to 10 μm,preferably 1 to 5 μm, more preferably 1 to 2 μm.

“nanopartide”: As used herein, “nanoparticle” refers to syntheticparticles of controlled dimension in the order of nanometers, whereinpreferably ISS-NA can be entrapped, encapsulated, non-covalently bound,covalently attached or dissolved in said nanoparticles. The size of ananoparticle is preferably less than 1000, 900, 800, 700, 600, 500, 400,300, 200, 100 or 50 nm, wherein further preferably said nanoparticle isnot smaller than about 10, 15, 20, 30, 40, or 50 nm. Preferably thenanoparticle is not smaller than 50 nm. Thus, nanoparticles of theinvention are preferably 10 to 500 nm, more preferably 20 to 400 nm,still more preferably 40 to 300 nm, still more preferably 50 to 200 nmand most preferably 50 to 100 nm in size. Preferred in the context ofthe invention are nanoparticles of 100 to 300 nm in size, more preferredare nanoparticles of 100 to 200 nm in size. Very preferred arenanoparticles of 50 to 200 nm in size. The term nanoparticle encompassesparticles of spherical, elliptic, longitudinal or irregular structure,preferably comprising or consisting of at least one a polymer.Nanoparticle also encompasses nanocapsules, and poliplex particles.Nanocapsules are nanoparticles comprising a reservoir, e.g. oilyreservoir, wherein said reservoir is surrounded by a polymer wall (MariaJ. Alonso, in Microparticulate systems for the delivery of proteins andvaccines. Eds. S. Cohen and Howard Bernstein, Marcel Dekker, New York1996, p206). A nanoparticle of a certain material, e.g. a polyesternanoparticle, refers to a nanoparticle comprising or preferablyessentially consisting, most preferably consisting of said material. Inthe context of the invention this formulation does not exclude thepresence of the ISS-NA in the nanoparticle.

Polymers suitable for the production of nanoparticles, includingnanocapsules, refer to: biodegradable polyesters (e.g. poly-lactic acid,poly-(lactic glycolic acid) copolymer (referred to as PLA and PLGA),poly-ε-caprolactone (referred to as PECL)), poly-(alkylcyanoacrylates)(referred to as PACA), chitosan, alginate, cross-linked human serumalbumin, human serum albumin, gelatin, Schizofylian, dextran.Nanoparticles may also be prepared from non-biodegradable materials suchas polystyrene, colloidal gold, silica, or metal clusters. In addition,hydrophilic components such as e.g. PEG, polysaccharides (Lemarchand C.et al. (Eur J Pharm Biopharm. 2004 September; 58:327-41), dextran,chitosan, polylysine, lecithine and the like may also be incorporated ascopolymer or as coating reagent on the surface of the nanoparticle.Complexation agent such as polylysine (PLL), poly(ethylene imine) (PEI),protamine, spermine or positively charged structured oligopeptides maybe incorporated into nanoparticles together with nucleic acids, bound tothe surface of nanoparticles for incorporation of nucleic acids,covalently attached to reactive groups on the nanoparticle, orincorporated during the polymerization process into the nanoparticle.Alternative complexation agents include metal salts such as Zincacetate.

“polyplex”: Polyplex particles are nanoparticles formed by the directinteraction of a cationic polymer, also called polycation, suchpolylysine (PLL), poly(ethylene imine) (PEI) or the like with a nucleicacid, preferably an ISS-NA. A Polyplex may also be formed by the directinteraction of PEG-PLL, or, for example, of a branched PEI with saidnucleic acid, preferably said ISS-NA.

“Complexation agent”: a complexation agent is an agent whichnon-covalently binds to an ISS-Na and neutralizes or at least partiallyreduces the effective charge of the resulting complex. Examples ofcomplexation agents are polylysine (PLL), spermine, protamine,poyethyleneimine (PEI), branched PEI, lysine-rich structuredoligopeptides, cationic lipids such as DOTAP and the like, and metalcations as provided by their salts such as zinc acetate, magnesium,calcium chloride, or the like. More complex polymers, such aspoly(ethylene)glycol-polylysine (PEG-PLL) copolymers may also be used ascomplexation agent.

“biodegradable”: A material, preferably a particle, most preferably amicroparticle or nanoparticle, is referred to as biodegradable when itis degradable or erodable under normal mammalian physiologicalconditions. Degradation of the particle may occur, for example, bydissolving of the particle, by enzymatic degradation, preferably byhydrolysis or oxidation, or by destabilisation of the particle by anyother chemical or physical process. Normal mammalian physiologicalconditions can be recreated in the test-tube by incubating samples inserum at 37° C. Microparticles or nanoparticles are consideredbiodegradable, if they are degraded upon incubation for 72 hours at 37°C. in human serum from healthy volunteers. In the context of thisdefinition, “degraded” means that the microparticle or nanoparticleloses at least 5%, preferably at least 10%, more preferably at least20%, still more preferably at least 50% of its mass and/or averagepolymer length. Most preferably, the particle is completely degradedunder these conditions. Conversely, a microparticle or nanoparticle isreferred to as non-biodegradable, if it does not loses at least 5% ofits mass and/or average polymer length upon incubation in human serumfor 72 hours at 37° C. Very preferred are particles which arebiodegradable at low pH, preferably at a pH which is found in theendosomes of immune cells, more preferably at ph of 4.5 to 6, mostpreferably at pH of about 5.

“coat protein”: As used herein, the term “coat protein(s)” refers to theprotein(s) of a bacteriophage or a RNA bacteriophage capable of beingincorporated within the capsid assembly of the bacteriophage or the RNAbacteriophage. However, when referring to the specific gene product ofthe coat protein gene of RNA bacteriophages the term “CP” is used. Forexample, the specific gene product of the coat protein gene of RNAbacteriophage Qβ is referred to as “Qβ CP”, whereas the “coat proteins”of bacteriophage Qβ comprise the “Qβ CP” as well as the A1 protein. Thecapsid of Bacteriophage Qβ is composed mainly of the Qβ CP, with a minorcontent of the A1 protein. Likewise, the VLP Qβ coat protein containsmainly Qβ CP, with a minor content of A1 protein.

“recombinant VLP”: The term “recombinant VLP”, as used herein, refers toa VLP that is obtained by a process which comprises at least one step ofrecombinant DNA technology. The term “VLP recombinantly produced”, asused herein, refers to a VLP that is obtained by a process whichcomprises at least one step of recombinant DNA technology. Thus, theterms “recombinant VLP” and “VLP recombinantly produced” areinterchangeably used herein and should have the identical meaning.

“virus particle”: The term “virus particle” as used herein refers to themorphological form of a virus. In some virus types it comprises a genomesurrounded by a protein capsid; others have additional structures (e.g.,envelopes, tails, etc.).

“virus-like particle (VLP)”, as used herein, refers to a non-replicativeor non-infectious, preferably a non-replicative and non-infectious virusparticle, or refers to a non-replicative or non-infectious, preferably anon-replicative and non-infectious structure resembling a virusparticle, preferably a capsid of a virus. The term “non-replicative”, asused herein, refers to being incapable of replicating the genomecomprised by the VIP. The term “non-infectious”, as used herein, refersto being incapable of entering the host cell. Preferably a virus-likeparticle in accordance with the invention is non-replicative and/ornon-infectious since it lacks all or part of the viral genome or genomefunction. In one embodiment, a virus-like particle is a virus particle,in which the viral genome has been physically or chemically inactivated,removed by disassembly and reassembly, or by assembly of purifiedproteins into a VLP. Typically and more preferably a virus-like particlelacks all or part of the replicative and infectious components of theviral genome. A virus-like particle in accordance with the invention maycontain nucleic acid distinct from their genuine. A typical andpreferred embodiment of a virus-like particle in accordance with thepresent invention is a viral capsid such as the viral capsid of thecorresponding virus, bacteriophage, preferably RNA bacteriophage. Theterms “viral capsid” or “capsid”, refer to a macromolecular assemblycomposed of viral protein subunits. Typically, there are 60, 120, 180,240, 300, 360 and more than 360 viral protein subunits. Typically andpreferably, the interactions of these subunits lead to the formation ofviral capsid or viral-capsid like structure with an inherent repetitiveorganization, wherein said structure is, typically, spherical ortubular. For example, the capsids of RNA bacteriophages or HBcAgs have aspherical form of icosahedral symmetry. The term “capsid-like structure”as used herein, refers to a macromolecular assembly composed of viralprotein subunits resembling the capsid morphology in the above definedsense but deviating from the typical symmetrical assembly whilemaintaining a sufficient degree of order and repetitiveness. Theinvention encompasses VLPs, preferably non-natural VLPs, comprising aicosahedral symmetry. One common feature of virus particle andvirus-like particle is its highly ordered and repetitive arrangement ofits subunits.

“virus-like particle of a RNA bacteriophage”: As used herein, the term“virus-like particle of a RNA bacteriophage” refers to a virus-likeparticle comprising, or preferably consisting essentially of orconsisting of coat proteins, mutants or fragments thereof, of a RNAbacteriophage. In addition, virus-like particle of a RNA bacteriophageresembling the structure of a RNA bacteriophage, being non replicativeand/or non-infectious, and lacking at least the gene or genes encodingfor the replication machinery of the RNA bacteriophage, and typicallyalso lacking the gene or genes encoding the protein or proteinsresponsible for viral attachment to or entry into the host. Thisdefinition should, however, also encompass virus-like particles of RNAbacteriophages, in which the aforementioned gene or genes are stillpresent but inactive, and, therefore, also leading to non-replicativeand/or non-infectious virus-like particles of a RNA bacteriophage.Preferred VLPs derived from RNA bacteriophages exhibit icosahedralsymmetry and consist of 180 subunits. Within this present disclosure theterm “subunit” and “monomer” are interexchangeably and equivalently usedwithin this context. Preferred methods to render a virus-like particleof a RNA bacteriophage non replicative and/or non-infectious is byphysical, chemical inactivation, such as UV irradiation, formaldehydetreatment, typically and preferably by genetic manipulation.Alternatively, individual proteins may be isolated from whole virionsand assembled into VLPs in vitro.

“synthetic VLP”: Particles formed from non-naturally occurring proteinsor peptides which spontaneously assemble intracellularly or in vitro arereferred to as synthetic VLPs. Examples for synthetic VLPs are providedin WO4071493A1 which is incorporated herein by reference. Synthetic VLPsalso encompasses particles which require nucleic acids or metal ions forassembly. Preferred synthetic particles have a defined size of 10 to1000 nm, preferably 10 to 300 nm, more preferably 10 to 150 nm, and mostpreferably 15 to 100 nm. Other preferred synthetic VLPs may exist in twoor more defined conformations, e.g. 15 nm and 25 nm.

“virosome”: As used herein, the term virosome relates to a reconstitutedvirus envelope, preferably to a reconstituted influenza virus envelope,more preferably to a reconstituted envelope of influenza A virus, mostpreferably to a reconstituted envelope of influenza A/Singapore virus.Virosomes are known in the art as drug carrier systems. A virosomecomprises a lipid membrane, wherein said lipid membrane typically andpreferably comprises a unilamellar lipid bilayer. In a preferred meaningthe term virosome relates to a reconstituted influenza virus envelope,preferably to a reconstituted influenza A virus envelope, mostpreferably to a reconstituted influenza A/Singapore virus envelope,wherein said reconstituted influenza virus envelope, preferably saidreconstituted influenza A virus envelope, most preferably saidreconstituted influenza A/Singapore virus envelope comprises lipidmembrane, wherein said lipid membrane comprises influenza glycoproteins,wherein preferably said influenza glycoproteins are selected fromhemagglutinin HA and neuraminidase NA. Typically and preferablyvirosomes attach to target cells via HA. Very preferred virosomescomprise a lipid membrane, preferably a lipid bilayer, wherein saidlipid membrane or said lipid bilayer comprise or preferablypredominantly consist of cationic lipids. Virosomes comprising cationiclipids in their lipid membrane are particularly suited for the deliveryof ISS-NA, especially of oligonucleotides. Further preferred arespecific virosomes, which attach to a target cell by an antibody or afragment thereof which is comprised in the lipid membrane. Thus, in afurther preferred meaning, the term virosome relates to a specificvirosomes comprising a lipid membrane, wherein said lipid membranecomprises specific antibodies or fragments thereof, preferably Fabfragments or Fab′ fragments, wherein preferably the specificity of saidantibodies or fragments thereof allows to direct the virosome to aspecific target cell. Typically and preferably, a virosome is taken upby the target cell by receptor-mediated endocytosis.

“effective amount”: As used herein, the term “effective amount” refersto an amount necessary or sufficient to realize a desired biologiceffect. An effective amount of the composition, preferably thepharmaceutical composition, would be the amount that achieves thisselected result, and such an amount could be determined as a matter ofroutine by a person skilled in the art. For example, an effective amountfor treating an immune system deficiency could be that amount necessaryto cause activation of the immune system, resulting in the developmentof an antigen specific immune response upon exposure to antigen. Theterm is also synonymous with “sufficient amount”. The effective amountfor any particular application can vary depending on such factors as thedisease or condition being treated, the particular composition beingadministered, the size of the subject, and/or the severity of thedisease or condition. One of ordinary skill in the art can empiricallydetermine the effective amount of a particular composition of thepresent invention without necessitating undue experimentation.

“treatment”: As used herein, the terms “treatment”, “treat”, “treated”or “treating” refer to prophylaxis and/or therapy. When used withrespect to an atopic disease, for example, the term refers to atherapeutic treatment which reduces the symptom score of the treatedpatient as assessed by a standard method commonly used to assess theseverity of the symptoms of the disease the patient is suffering from.The term can also refer to a prophylactic treatment of an atopic diseasewhich, for example, prevents or ameliorates the symptoms a patienttypically develops upon exposure to a challenging agent as compared toan untreated patient.

“pharmaceutically acceptable carrier”: The compositions of the inventioncan be combined, optionally, with a pharmaceutically-acceptable carrier.The term “pharmaceutically-acceptable carrier” as used herein means oneor inure compatible solid or liquid fillers, diluents or encapsulatingsubstances which are suitable for administration into a human or otheranimal. The term “carrier” denotes an organic or inorganic ingredient,natural or synthetic, with which the active ingredient is combined tofacilitate the application.

“pharmaceutical composition”: As used herein, the term “pharmaceuticalcomposition” refers to a formulation which contains the composition ofthe invention and which is in a form that is capable of beingadministered to an animal. Typically and preferably, the pharmaceuticalcomposition comprises a conventional saline or buffered aqueous solutionmedium in which the composition of the present invention is suspended ordissolved. In this form, the composition of the present invention can beused conveniently to prevent, ameliorate, or otherwise treat acondition. Upon introduction into a host, the pharmaceutical compositionis able to provoke an immune response including, but not limited to, theproduction of antibodies and/or cytokines and/or the activation ofcytotoxic T cells, antigen presenting cells, helper T cells, dendriticcells and/or other cellular responses. Preferred pharmaceuticalcompositions induce a cytokine milieu, typically and preferably theformation of IFN-alpha, which is reducing allergic and/or asthmaticsymptoms. Optionally, the pharmaceutical composition additionallyincludes an adjuvant which can be present in either a minor or majorproportion relative to the composition of the present invention.

“adjuvant”: The term “adjuvant” as used herein refers to non-specificstimulators of the immune response or substances that allow generationof a depot in the host which when combined with the composition of theinvention provides for an even more enhanced and/or prolonged immuneresponse, preferably cytokine production. A variety of adjuvants isknown in the art and useful in the invention. Preferred adjuvants areselected from the group consisting of incomplete Freund's adjuvant,aluminum containing adjuvants, modified muramyldipeptide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, BCG (bacilleCalmette Guerin) Corynebacterium parvum, ligands of toll-like receptors(TLR) which include but are not limited to peptidoglycans,lipopolysaccharides and their derivatives, poly I:C, immunostimulatoryoligonucleotides, imidazoquinolines such as resiquimod and imiquimod,flagellins, monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21,QS-18, GPI-0100, CRL1005, MF-59, OM-174, OM-197, OM-294, Virosomaladjuvant technology and any mixture thereof. A very preferred adjuvantfor the purpose of the invention is aluminium containing adjuvant,preferably an aluminium containing mineral gel, most preferablyalhydrogel. In a very preferred embodiment said adjuvant is alhydrogel.The term adjuvant also encompasses a mixture of any of the substanceslisted above. Particles of the invention, preferably VLPs, have beengenerally described as an adjuvant. However, the term “adjuvant”, asused within the context of this application, refers to an adjuvant notbeing the particle of the invention, in particular not the VLP used forthe inventive compositions. In each case, the term adjuvant refers to anadjuvant used in addition to said particle.

“polypeptide”: As used herein the term “polypeptide” refers to a polymercomposed of amino acid residues, generally natural amino acid residues,linked together through peptide bonds. Although a polypeptide may notnecessarily be limited in size, the term polypeptide is often used inconjunction with peptide of a size of about ten to about 50 amino acids.

“protein”: As used herein, the term protein refers to a polypeptide of asize of above 20, more preferably of above 50 amino acid residues.Proteins generally have a defined three dimensional structure althoughthey do not necessarily need to, and are often referred to as folded, incontrast to peptides and polypeptides which often do not possess adefined three-dimensional structure, but rather can adopt a large numberof different conformations, and are referred to as unfolded.

“sequence identity”: The amino acid sequence identity of polypeptidescan be determined conventionally using known computer programs such asthe Bestfit program. When using Bestfit or any other sequence alignmentprogram, preferably using Bestfit, to determine whether a particularsequence is, for instance. 95% identical to a reference amino acidsequence, the parameters are set such that the percentage of identity iscalculated over the full length of the reference amino acid sequence andthat gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed. This aforementionedmethod in determining the percentage of identity between polypeptides isapplicable to all proteins, polypeptides or a fragment thereof disclosedin this invention.

“Sequence homology”: The homology of nucleotide sequences is preferablydetermined by the program blastn which is an implementation of the BLASTalgorithm, most preferably using the default settings of the software.

“fragment of a protein”, in particular fragment of a recombinant proteinor recombinant coat protein, as used herein, is defined as apolypeptide, which is of at least 70%, preferably at least 80%, morepreferably at least 90%, even more preferably at least 95% the length ofthe wild-type recombinant protein, or coat protein, respectively andwhich preferably retains the capability of forming VLP. Preferably thefragment is obtained by at least one internal deletion, at least onetruncation or at least one combination thereof. The term “fragment of arecombinant protein” or “fragment of a coat protein” shall furtherencompass polypeptide, which has at least 80%, preferably 90%, even morepreferably 95% amino acid sequence identity with the “fragment of arecombinant protein” or “fragment of a coat protein”, respectively, asdefined above and which is preferably capable of assembling into avirus-like particle.

The term “mutant coat protein” refers to a polypeptide having an aminoacid sequence derived from the wild type recombinant protein, or coatprotein, respectively, wherein the amino acid sequence is at least 80%,preferably at least 85%, 90%, 95%, 97%, or 99% identical to the wildtype sequence and preferably retains the ability to assemble into a VLP.

“animal”: As used herein, the term “animal” refers to any animal,preferably to any animal comprising an immune system, includingnon-vertebrates, preferably arachnids and insects, and vertebrates.Typically and preferably, animal relates to vertebrates, more preferablyto mammals, most preferably to humans. Thus, animal includes, forexample, humans, sheep, horses, cattle, pigs, dogs, cats, rats, mice,birds, reptiles, fish, insects and arachnids.

“one”, “a/an”: When the terms “one,” “a,” or “an” are used in thisdisclosure, they mean “at least one” or “one or more,” unless otherwiseindicated.

“about”: within the meaning of the present application the expressionabout shall have the meaning of +/−10%. For example about 100 shall mean90 to 110.

As previously disclosed, VLPs packaged with unmethylated CpG-containingoligonucleotides can enhance B and T cell responses. It was alsopreviously observed, that application of a CpG-containingoligonucleotide together with an allergen ameliorated allergic response.It was now surprisingly found that no covalent linkage, coupling ormixing of allergen with the particle of the invention, preferably theVLP, is required to achieve an effect in the treatment ofhypersensitivity, preferably allergy. Even more surprisingly, it wasfound that no administration or co-administration of a specific antigenor allergen is required in the treatment of hypersensitivity, preferablyallergy, using the compositions of the invention. Furthermore, saideffect is not limited to VLPs packaged with unmethylated CpG-containingoligonucleotides but can be generalized to particles of the inventionpackaged with immunostimulatory nucleic acids (ISS-NA).

Applicants have discovered that hypersensitivities may be cured bytreatment with particles packaged with ISS-NA. Applicants have alsoshown that particles of appropriate size diffuse readily to draininglymph nodes after intradermal or subcutaneous injection, where they aretaken up mainly by dendritic cells (DC) and macrophages. Various DCs,including plasmacytoid dendritic cells (pDC), monocyte dreived andleucophil dendritic cells or macrophages are activated upon uptake ofparticles, preferably VLPs, packaged with unmethylated CpG-containingoligonucleotide via the Toll-9 receptor. Applicants have also discoveredthat polystyrene nanoparticles with sizes of 50 to 200 nm also readilydiffuse to the draining lymph node, while larger particles (>500 nm)remain at the site of injection during the same time span aftersubcutaneous injection. PLGA nanoparticles have been shown to bephagocytosed by dendritic cells (DCs) (Diwan et al. J Drug Target. 2003;11:495-507). Moreover murine DCs pulsed with PLGA nanoparticles havingco-incorporated an unmethylated CpG-containing oligodesoxynucleotide andan antigen have been shown to enhance antigen specific T-cell activationin an in vitro T-cell stimulation assay, as compared to DCs pulsed withPLOA nanoparticles incorporating only antigen. Therefore, nanoparticlesincorporating an ISS-NA like an unmethylated CpG-containingoligodesoxynucleotide are able to stimulate dendritic cells upon uptake,and these dendritic cells thereafter activated T-cells. In humandendritic cells, Toll-like receptor 9 expression is limited to a smallsubset, the so called plasmacytoid dendritic cells. We show herein thatQβ virus-like particles, which have a size of about 30 nm, are taken upby human plasmacytoid dendritic cells, and therefore can deliver ISS-NAto these cells. Interestingly, mast cells also express TLR-9. A recentreport has shown the role of CpG-containing oligonucleotide inpreventing Th2-cell activation upon allergen challenge (Hessel et al.(2005) J. exp. Med. 11:1563-73.). Therefore, uptake of particles such asnanoparticles or VLPs packaged with ISS-NA, such as unmethylatedCpG-containing oligonucleotide, by plasmacytoid dendritic cells orantigen presenting cells such as classical dendritic cells may lead toinhibition of the activation of Th2 cells and thereby inhibit theallergen induced response. The same report involved mast cells in thesuppressive action of unmethylated CpG-containing oligonucleotide on theallergic response. Mast cells are active in phagocytosis, and it ispossible that particles of the invention, preferably nanoparticles orVLPs, wherein said nanoparticles or VLPs, preferably said VLPs, arepackaged with ISS-NA, preferably with unmethylated CpG-containingoligonucleotide, mediate their effect in part via inhibiting mast cells,providing for another alternative or complementary possible mechanism ofaction of the VLP or nanoparticle packaged with unmethylatedCpG-containing oligonucleotide. However, the mode of action of thecompositions of the invention is not limited to this mechanism.

Based on the aforementioned findings, the invention providescompositions, pharmaceutical compositions and methods for the treatmentor prevention of hypersensitivity, preferably allergy in an animal. Inparticular, the invention provides a composition for use in a method oftreating hypersensitivity in an animal, the composition comprising aparticle and an ISS-NA, wherein said particle is packaged with saidISS-NA. The invention provides methods and compositions which areespecially useful for treating and/or preventing hypersensitivity,preferably allergy, more preferably atopic eczema, atopic asthma andtype I allergies like, for example, pollen allergy (hay fever).

In this context, the term particle refers to any structure which, withrespect to its chemical an physical characteristics, can be packagedwith ISS-NA, wherein preferably said particle is capable of protectingsaid ISS-NA from degradation in the body of said animal and/or whereinsaid particle is capable of specifically delivering and releasing saidISS-NA to immune cells. Thus, to be effective, the particle of theinvention preferably is able to (i) package an ISS-NA and (ii) todeliver said ISS-NA to immune cells in the body. The particle of theinvention is therefore to be understood in a very broad sense.

In a preferred embodiment said particle is selected from the groupconsisting of synthetic particle, virus particle and VLP.

The particles of the invention can be biodegradable or nonbiodegradable. Biodegradable particles are generally preferred toprevent accumulation of said particle in the body of said animal and toavoid toxicity effects which might be associated with such accumulation.Therefore, in a preferred embodiment said particle, preferably saidsynthetic particle, most preferably said nanoparticle, comprises or,preferably, essentially consists of, most preferably consists of abiodegradable material. In a further preferred embodiment said particle,preferably said synthetic particle, most preferably said nanoparticle,is biodegradable. In a very preferred embodiment said synthetic particlepackaged with said ISS-NA is biodegradable. In a further preferredembodiment said particle, preferably said synthetic particle, releasessaid ISS-NA, preferably inside the target cell, most preferably in theendosome of the target cell. In a further preferred embodiment saidparticle, preferably said synthetic particle is degraded in theendosomal compartment of the target cell containing proteases andexhibiting a low pH, preferably about pH 5.

In one embodiment of the invention said particle is a syntheticparticle, preferably a synthetic particle selected from the groupconsisting of liposome, microparticle and nanoparticle, wherein morepreferably said synthetic particle is liposome or a nanoparticle, andeven more preferably a nanoparticle. In a further embodiment of theinvention said particle is a synthetic particle, preferably a syntheticparticle selected from the group consisting of liposome, microparticle,nanoparticle and virosome. Synthetic particles of the invention compriseor preferably essentially consist of, most preferably consist ofnon-biodegradable materials, of biodegradable materials or of a mixtureof both, wherein said biodegradable materials may be organic orinorganic, or a combination of both.

In a preferred embodiment said synthetic particle is a microparticle ora nanoparticle, preferably a nanoparticle, wherein said syntheticparticle comprises or preferably essentially consists of, mostpreferably consists of a material selected from the group consisting of:(a) polyesters, preferably selected from PLA, poly(glycolic acid) andPECL, (b) copolymers of polyesters, preferably PLGA, (c) blockcopolymers of polyester and PEG, (d) polyorthoesters, (e)poly(anhydrides), (f) poly(sebacic acid), (g) polyanhydrides based onsebacic acid monomers incorporating amino acids, (h) polyanhydrideesters, (i) polyphosphazene, preferably polyphosphazene containinghydrolysis-sensitive ester groups (Andrianov A K and Payne L G, inMicroparticulate systems for the delivery of proteins and vaccines, Eds.S. Cohen and Howard Bernstein, Marcel Dekker, New York 1996, p127-147),(j) polyamide, (k) macromolecules and modified macromolecules ofbiological origins, preferably selected from proteins (e.g. gelatin,human serum albumin) and (l) polysaccharides (e.g. dextran, chitosan,alginate, Schyzofyllan), (m) methacrylate-based materials, preferablyselected from poly(methyl methacrylate) and copolymers, preferablyPEG-methacrylate or PEG-dimethacrylate, (n) methyl methacrylate basedmaterials, preferably selected from PEG based comonomers and ioniccomonomers, (O) poly(methylidene malonate 2.1.2) as described byBousquet Y et al. Biomaterials. 1998 January-February; 19:271-8 andBreton P et al. Pharm Res. 1999; 16:141-7, (p) colloidal gold, (q)polystyrene, (r) polyethylene, (s) polypropylene, (t) latex, (u)ferromagnetic or paramagnetic materials, (v) dextran, (w)hydroxyapatite, and (x) a mixture of any of the materials listed above.

In a preferred embodiment said synthetic particle is a microparticle ora nanoparticle, preferably a nanoparticle, wherein said syntheticparticle comprise or preferably essentially consist of, most preferablyconsist of a non-biodegradable material selected from the groupconsisting of: (a) colloidal gold, (b) polystyrene. (c) polyethylene,(d) polypropylene, (e) latex, (f) ferromagnetic or paramagneticmaterials, (g) dextran and (h) hydroxyapatite.

In a more preferred embodiment said synthetic particle, preferably saidnanoparticle, comprises or preferably essentially consists of, mostpreferably consists of a biodegradable material selected from the groupconsisting of: (a) polyesters, preferably selected from PLA,poly(glycolic acid) and PECL, (b) copolymers of polyesters, preferablyPLGA, (c) block copolymers of polyester and PEG, (d) polyorthoesters,(e) poly(anhydrides), (f) poly(sebacic acid), (g) polyanhydrides basedon sebacic acid monomers incorporating amino acids, (h) polyanhydrideesters, (i) polyphosphazene, preferably polyphosphazene containinghydrolysis-sensitive ester groups (Andrianov A. K. and Payne L G, inMicroparticulate systems for the delivery of proteins and vaccines, Eds.S. Cohen and Howard Bernstein, Marcel Dekker, New York 1996, p.127-147), (j) polyamide, and (k) macromolecules and modifiedmacromolecules of biological origins, preferably selected from proteins(e.g. gelatin, human serum albumin) and polysaccharides (e.g. dextran,chitosan, alginate, Schyzofyllan).

In a still more preferred embodiment said biodegradable material isselected from (a) polyesters, preferably selected from PLA,poly(glycolic acid) and PECL, (b) copolymers of polyesters, preferablyPLGA, (c) block copolymers of polyester and PEG, (d) polyorthoesters,(e) poly(anhydrides), (f) poly(sebacic acid), (g) polyanhydrides basedon sebacic acid monomers incorporating amino acids, (h) polyanhydrideesters, (i) polyphosphazene, preferably polyphosphazene containinghydrolysis-sensitive ester groups (Andrianov A K and Payne L G, inMicroparticulate systems for the delivery of proteins and vaccines, Eds.S. Cohen and Howard Bernstein, Marcel Dekker, New York 1996, p.127-147), and (j) polyamide.

In a still more preferred embodiment said biodegradable material isselected from (a) polyesters, preferably polyesters selected from PLA,poly(glycolic acid) and PECL, and (b) copolymers of polyesters,preferably PLGA. In a very preferred embodiment said biodegradablematerial is PLA or PLGA, most preferably PLA.

In a further preferred embodiment said particles, preferably saidsynthetic particles, most preferably said nanoparticles comprise orpreferably essentially consist of at least one, preferably exactlythree, more preferably exactly two, most preferably exactly onebiodegradable polymer(s), wherein preferably said polymer is degradedinto biocompatible non-toxic monomers, wherein still more preferablysaid biodegradable polymer is a polyester (see Maria J. Alonso et al.,in Microparticulate systems for the delivery of proteins and vaccines,Eds. S. Cohen and Howard Bernstein, Marcel Dekker, New York 1996, p203-242).

Nanoparticles may be formed by chemical or physical processes such aspolymerization or condensation of monomers, for example in nanodropletsof an emulsion, precipitation of polymers, coacervation ofmacromolecules, assembly of macromolecules brought together, forexample, by the process of emulsification, by ionic interactions,aggregation, heat denaturation or chemical cross-linking of saidassembled macromolecules. Nanoparticles may also have a compositestructure involving two or more layers with different properties, suchas core-shell particles with a hydrophobic core layer of a polymer and ahydrophilic outer shell layer of another polymer, such aspoly(ethylene)glycol (PEG). Other examples of core-shell nanoparticlesinclude nanoparticles where a charged polymer, such as polylysine, isadsorbed to the surface of the nanoparticle. Alternatively, more complexnanoparticles may be produced, where co-monomers carrying a cationicmoiety and co-monomers carrying a PEG moiety are included in thepolymerization process and preferentially locate to the nanoparticlesurface during the polymerization process. Thus, in a preferredembodiment said nanoparticle comprises an outer shell layer, whereinpreferably said outer shell layer comprises or alternatively essentiallyconsists of PEG. In a very preferred embodiment said nanoparticlecomprises a core and a shell layer, wherein said core comprises oralternatively essentially consists of polyester and wherein said shelllayer comprises or alternatively essentially consists of PEG. In afurther preferred embodiment said nanoparticle comprises a core and ashell layer, wherein said core comprises or alternatively essentiallyconsists of polyalkylcyanoacrylat and wherein preferably said shelllayer comprises or alternatively essentially consists of PEG.

In a further preferred embodiment said nanoparticle is a core-shellnanoparticle, wherein preferably the shell layer of said core-shellnanoparticle is packages with said ISS-NA. Preferably, said shell layerof said core-shell nanoparticle is positively charged. Furtherpreferably said shell layer of said core-shell particle comprises oralternatively essentially consists of a polymer, wherein preferably saidpolymer is selected from polylysine, cethyltrimethylammonium bromide(CTAB) and polyethyleneimine. Alternatively, said ISS-NA maypreferentially associate with the core layer, for example of aCaP-PEG-PAA nanoparticle.

In a further preferred embodiment said nanoparticle is a polyplex. Apolyplex nanoparticle is formed by the direct association of acomplexation agent such as PEI, PLL or PEG-PLL with an ISS-NA, forming aso called polyplex (Boeckle S. et al. J Gene Med 2004; 6:1102-1111;Wagner E. et al. Adv Genet. 2005; 53:333-54; Walker G. et al. Mol Ther.2005; 11:418-25).

The size of nanoparticles can be determined with known methods such asscanning- and transmission-electron microscopy, or photon correlationspectroscopy or dynamic light scattering, as reviewed in Alonso M J. (inMicroparticulate systems for the delivery of proteins and vaccines, Eds.S. Cohen and Howard Bernstein, Marcel Dekker, New York 1996, p203-242).Surface properties such as surface charge, and in particular thezeta-potential, can be measured by determining the velocities ofnanoparticles in an electrical field using methods such aselectrophoretic mobility measurement. Laser Doppler anemometry orElectrophoretic light scattering may also be used. Alternatively, thesonic response to an alternating electric field (electronic sonicamplitude effect) may be used as well. The measurements can be performedwith commercially available devices. The zeta potential is informativeto predict the aggregation behaviour of a nanoparticle, or its abilityto adsorb charged components on its surface, and thus helps optimizingthe properties of nanoparticles.

In a preferred embodiment said nanoparticle comprises or alternativelyessentially consists of polyesters, preferably of aliphatic polyesters.Aliphatic polyesters are degraded by random hydrolytic cleavage, intophysiologically occurring metabolites. These materials have been usedfor resorbable sutures, and a poyester microsphere formulation forparenteral injection of Leuprolide acetate, a Gonadotropin ReleasingHormone analog, is a marketed product. Several polymers can be used inthe preparation of the nanoparticles of the invention and have beendescribed: poly(lactic acid) (PLA), poly(glycolic acid) (PGA), orCopolymers of PLAA and PGA, poly(-lactic-co-glycolic acid) named PLGA,are particularly suited. The racemic, amorphous form is preferred. Thesepolymers are commercially available, and their synthesis is well knownin the art. Their synthesis is described, for example, by Avgoustakis K(Curr Drug Deliv. 2004; 1:321-33). The properties of polyestermicrosphere, and PLGA in particular, as well as methods to produce themhave been reviewed and described for example by Kissel T and Koneberg R(in Microparticulate systems for the delivery of proteins and vaccines,Eds. S. Cohen and Howard Bernstein, Marcel Dekker, New York 1996, p.51-87). These methods include spray drying methods,water-in-oil-in-water emulsion solvent evaporation methods and phaseseparation methods. In a preferred embodiment said nanoparticlecomprises or alternatively essentially consists of PLGA, whereinpreferably the molar ratio of the monomers constituting PLGA is 50% molLA and 50% mol GA. Increasing the proportion of either of the monomerleads to slower degradation of the polymer. For example, a polymer witha ratio of LA to GA of 85:15 has a rate of degradation about two- and ahalf time slower than with a ratio of 50:50. Thus, the porperties ofPLGA polymer can be manipulated by changing the proportion of themonomers.

Preparation of nanoparticles and in particular of nanoparticles madefrom polyester materials has been reviewed for example by Alonso M J,(in Microparticulate systems for the delivery of proteins and vaccines,Eds. S. Cohen and Howard Bernstein, Marcel Dekker, New York 1996,p203-242) or Avgoustakis K (Curr Drug Deliv. 2004; 1:321-33). Methodsfor producing polyester nanoparticles are also described therein, andinclude methods collectively referred to as polymer precipitationmethods. In these methods, the polymer dissolved in an organic solventis emulsified in water containing a stabilizer. The organic solventdiffuses from the organic phase into the aqueous phase. The result ofthe solvent depletion from the organic phase is polymer precipitation,leading to the formation of nanoparticles.

One of these is the solvent extraction-evaporation technique. In thismethod an organic solvent with poor water solubility, such as methylenechloride, ethyl acetate or chloroform is used. The solvent isnonetheless extracted from the organic phase into the water phase due tothe large excess of water, a process which is further facilitated bysubsequent evaporation of the solvent. The solventextraction-evaporation technique can be essentially performed in twogeneral ways to incorporate molecules into nanoparticles, depending onthe hydrophobicity of the molecule to be incorporated. A hydrophobicmolecule, or a water insoluble complex can be dissolved or suspended inthe same organic solvent as the polymer, and then be emulsified in anexternal water phase containing a stabilizer, typically a surfactantsuch as poly(vinyl alcohol) (PVA), a poloxamer, or for example dextran,whereby the nanodroplets formed during the emulsion process formnanoparticles upon solvent extraction and evaporation. Preferably, theemulsion is prepared using high-speed or high-pressure homogenizers,microfluidization or sonication. Intensive stirring or vortexing mayalso be appropriate. This method is also referred to as the oil-in-watersolvent extraction and evaporation technique.

More hydrophilic or amphiphilic compounds are incorporated as aqueoussolutions into a larger volume of an organic solvent containing thepolymer. Preferrably, the aqueous solution is emulsified with theorganic solvent, for example using high-speed or high-pressurehomogenizers, microfluidization, sonication, vortexing or intensivestirring. The resulting water-in-oil emulsion or suspension is furtheremulsified in a larger volume of water, yielding a water-in-oil-in wateremulsion, from which the nanoparticles form upon solvent extraction andevaporation. This method is also referred to as thewater-in-oil-in-water emulsion solvent evaporation technique.

The process of solvent extraction and evaporation can be furtheraccelerated for example by increasing the temperature, applying vacuumor adding an alcohol such as isopropanol (e.g. 2%) to the external waterphase to increase the organic solvent solubility in that external waterphase. For example, a rotavapor may be used to accelerate solventextraction and evaporation.

Some components such as certain surfactants used in the productionprocess and incompatible with parenteral injection into animals orhumans may have to be eliminated, for example by washing steps withwater. Purification techniques used are for example tangential-flowfiltration, or centrifugation. Nanoparticles are then typically isolatedby freeze-drying, whereby a cryoprotectant such as a sugar liketrehalose or glucose is added to prevent aggregation of thenanoparticles.

Another polymer precipitation technique is the solvent displacement ornanoprecipitation method. It involves the use of an organic solvent thatis completely soluble in the external water phase. The polymer isdissolved in acetone, ethanol or methanol, and precipitates uponincorporation under stirring into an aqueous solution of a surfactant,such as for example Poloxamer 188.

A further polymer precipitation method is the salting-out technique. Inthis method, the polymer is dissolved in, for example acetone, and asaturated aqueous solution of PVA is added under stirring to form anoil-in-water emulsion. Water is further added, and the polymerprecipitates to form nanoparticles when acetone diffuses into theexternal water phase.

In a further embodiment of the invention, said nanoparticle is aPEG-polyester nanoparticle, such as for example, PEG-PLA or PEG-PLGAnanoparticles. PLA and PLGA particles absorb plasma proteins such asopsonin, and activate the complement system. To minimize plasma proteinbinding, core-shell nanoparticles have been produced, where a layer of ahydrophilic PEG, the shell, surrounds the polymer core of the particle,and prevents or reduces attachments of plasma proteins or opsonin to thehydrophobic polymer surface. Coating of nanoparticles and in particularof PLGA nanoparticles with poly(ethylene glycol) has been shown tostongly reduce complement activation and increase blood circulation time(Gref R et al. in Microparticulate systems for the delivery of proteinsand vaccines, Eds. S. Cohen and Howard Bernstein, Marcel Dekker, NewYork 1996, p279-305; Hawley A E et al. Pharm Res. 1997; 14:657-61).Enrichment in the lymph nodes upon subcutaneous injection was alsoenriched compared to naked PLGA nanoparticles (Hawley A E et al. PharmRes. 1997; 14:657-61). When polystyrene or PLGA nanoparticles werecoated with PLA-PEG copolymer, maximal lymph node enrichment wasobtained with the shorter PEG chain (750 Da PEG, PLA:PEG 1.5:0.75;Hawley A E et al. Pharm Res. 1997; 14:657-61). Longer PEG chains led toincreased drainage from the injection site and lower lymph node levels,suggesting less efficient capture by lymph node macrophages and henceincreased systemic distribution as a result of the increased stericbarrier of the longer PEG chains. When PLA-PEG (PLA:PEG 1.5:0.75) wasincorporated in the PLGA nanoparticles during the precipitation process,the highest lymph node enrichment was obtained with nanoparticles havingan intermediate percentage of PLA-PEG (35%), while particles with 45%PLA-PEG had increased systemic distribution, as reflected by higherblood and liver levels. Thus, variation of the PEG chain length and ofthe percentage of the PEG copolymer incorporated in the nanoparticlesallows manipulation of the pharmacokinetic properties of thenanoparticles.

The methods for the preparation of PEG-PLA and PEG-PLGA nanoparticlesare similar to the methods used for the preparation of PLA and PLGAnanoparticles, and have been reviewed by Avgoustakis K (Curr Drug Deliv.2004; 1:321-33) and Gref R. et al. (in Micropaniculate systems for thedelivery of proteins and vaccines, Eds. S. Cohen and Howard Bernstein,Marcel Dekker, New York 1996, p279-305). These methods include theemulsion-solvent evaporation method, the solvent displacement method andthe salting-out method. Use of the salting-out method for thepreparation of nanocapsules is also described therein. Methods forsynthesizing block copolymers are know in the art. One method ofpreparing diblock copolymers (e.g. PEG-PLA) by ring-openingpolymerization of monomers (lactide, glycolide, caprolactone or mixturesof them) on momomethoxy-PEG catalyzed by stannous octoate, as well asthe preparation and use of multiblock copolymers for the preparation ofnanoparticles is described and reviewed by Avgoustakis K (Curr DrugDeliv. 2004:1:321-33) and references therein.

Variation of the PLA/PEG and PLGA/PEG allows to manipulate the structureof the nanoparticles produced. For example, a low ratio generates moredynamic type of particles, while higher ratios favour more solid-likestructures. A shorter PLA or PLGA chain length (×≦30) can lead tonanoparticle formation via micelle formation process, while with longerchain length an agglomeration-precipitation process is taking placeAvgoustakis K (Curr Drug Deliv. 2004; 1:321-33).

In a further embodiment of the invention, the nanoparticle is apolyalkylcyanoacrylate nanoparticle. Polyalkylcyanoacrylatenanoparticles may be prepared as described by Fattal E. et al. (J.Contr. Release 1998: 53:137-143), using an emulsion polymerizationprocess. The nanoparticles are then coated with a cationic hydrophobicreagent, such as cetyltrimethylammonium bromide (CTAB), and the ISS-NAis adsorbed on the nanoparticle. In an alternative method, such asdescribed by Zobel et al. (Antisense Nucleic Acid Drug Dev. 1997;7:483-493), where nanoparticles are produced in an aqueous dispersionpolymerization process. The surfactant used is DEAE-dextran, whichallows subsequent adsorption of ISS-NA on the surface of thenanoparticle through electrostatic interactions. Particles of sizesranging from about 170-1000 nm were obtained by Zobel et al.

In a further embodiment, the nanoparticle is a PEG-coatedpolycyanoacrylate nanoparticle, such as described by Li Y. et al. (IntJ. Pharm. 2003; 259:93-101), whereby the additional covalent attachmentof transferrin to the nanoparticles is omitted. The nanoparticles areprepared by a water-in-oil-in-water emulsion solvent evaporationtechnique, using poly(aminopoly(ethyleneglycol)-cyanoacrylate-co-hexadecyl cyanoacrylate)(poly(H₂NPEGCA-co-HDCA)) as polymer. Briefly, the ISS-NA diluted inbuffer (e.g. NaHCO₃, pH 8) is emulsified in dichloromethane/ethylacetate (1:1) containing poly(H₂NPEGCA-co-IIDCA) by sonication. Theresulting emulsion is poured into a 1% w/v PVA aqueous solution, andfurther emulsified. The percentage of PVA may be varied, and inparticular a higher percentage (3% in 0.1M NaHCO₃, pH 8) has been shownto limit damage to dsDNA (Li Y. et al. (Int J Pharm. 2003; 259:93-101).The resulting double emulsion is then diluted in a 0.3% w/v PVA aqueoussolution, under magnetic stirring. The organic solvents are theneliminated by evaporation under reduced pressure in a Rotavapor at 37°C., and the nanoparticles collected by centrifugation at 39000 g.Particle sizes obtained by Li et al. ranged from about 130 to 150 nm. Inone embodiment, the ISS-NA is an unmethylated CpG-containingoligonucleotide.

Packaging of ISS-NA, and in particular unmethylated CpG-containingoligonucleotide in a particle produced by an emulsion-solventevaporation technique or spray-dry technique, will be exemplified in thefollowing for polyester nanoparticles. However, it is apparent for theartisan that similar procedures can be applied for PACA and PEG-PACAnanoparticles. Packaging of ISS-NA, such as for example unmethylatedCpG-containing oligonucleotide, within polyester nanoparticles such asPLA, PLGA, PECL, PEG-PLA, PEG-PLGA or PEG-PECL nanoparticles, can beperformed in several ways. In one method, ISS-NA is added to the initialwater phase of the water-in-oil-in-water emulsion solvent evaporationtechnique, as has been reported by Aukunuru J. V. et al. (J. Pharm.Pharmacol. 2003; 55:1199-206). In another method, the ISS-NA issuspended in the organic solvent, e.g. dichloromethane, by sonication,and then spray dried to yield nanoparticles having packaged the ISS-NA.In certain embodiments, the capacity of a nanoparticle to package anISS-NA, and in particular an ODN, is increased by adding a complexationagent, such as lysine rich oligopeptide (Emile C. et al. Drug Deliv1996; 3:187-195), PLL, PEI, spermine or a salt such as Zinc acetate(Putney S D et al. Antisense Nucleic Acid Drug Dev. 1999; 9:451-8). Aninsoluble complex results from the mixing of the complexation agent andthe ISS-NA in an aqueous solution, which can be incorportated intonanoparticles. As would be understood, the optimal ratio of complexationagent to ISS-NA, and when the complexation agent is PEI or PLL, theoptimal length of the PLL or PEI, and when the complexation agent is PEIwhether branched or linear PEI is chosen, have to be determinedempirically. For example, a solution of the polymer, a complexationagent such as a lysine rich peptide, and of a ISS-NA such as an ODN, inacetone, is poured dropwise in an aqueous solution as reported by EmileC. et al. (Drug Deliv 1996; 3:187-195). The complex of the ISS-NA andthe complexation agent precipitates with the polymer to formnanoparticles packaged with the ISS-NA. In one embodiment, the complexof the ISS-NA and the complexation agent is suspended in an organicsolvent, e.g. methylene chloride, and incorporated and packaged in aPLGA nanoparticle using a spray-dry technique (Putney S D et al.Antisense Nucleic Acid Drug Dev. 1999; 9:451-8; Pamujula S et al. JPharm Phamracol 2004; 56:1119-25).

In a further embodiment said nanoparticle is a Block copolymer-coatedcalcium phosphate nanoparticle (CaP-PEG-PAA; Kakizawa et al. (2004) J.Contr. Release 97:345-56). These are core-shell particles suitable forpackaging ISS-NA, preferably unmethylated CpG-containingoligonucleotides. They comprise a core of nanocrystals of CaP,surrounded by a hydrophilic tethered layer of PEG. Mixing of a calcium-,and a phosphate-containing solution in the presence of ISS-NA andPEG-block-poly(aspartic acid) (PEG-PAA) leads to the spontaneousformation of nanoparticles incorporating ISS-NA. In one embodiment theISS-NA is an unmethylated CpG-containing oligonucleotide. ThePAA-segment of PEG-PAA has high binding affinity for CaP, and thenon-ionic and hydrophilic PEG has a steric stabilization function.Particle size can be adjusted by varying the PEG-PAA and phosphateconcentration, as seen in FIG. 1 of Kakizawa et al. (2004) J. Contr.Release 97:345-56. Typical sizes obtained are between about 100 to about300 nm. A critical minimal amount of PEG-PAA is required to preventaggregation of the nanoparticles, while it higher levels, it starts tocompete with ISS-NA for CaP binding, and optimal concentration andratios of the components have to be determined empirically as taught byKakizawa et al. Increasing phosphate concentration from 1.5 mM to 3 mM,for example, led to a higher ODN binding capacity (Kakizawa et al.(2004) J. Contr. Release 97:345-56).

Preparation of PEG-PAA and of the nanoparticles has been described indetail in (Kakizawa et al. (2004) J. Contr. Release 97:345-56). Inessence, a solution containing CaCl₂, Tris, a low EDTA amount and ISS-NAis added quickly to an equal volume of a solution containing Hepes,Disodium Hydrogen phosphate and PEG-PAA. The resulting mixture isvigorously stirred by a vortex mixer and incubated at 37° C. for 24hours. Kakizawa et al. envisage the use of Ca-P-PEG-PAA nanoparticlesfor delivery of siRNA to the cytoplasm, where they expect fastdissolution of the CaP core in the cytoplasm due to the lower Ca²⁺concentration and higher phosphate concentration. The ISS-NA of thepresent invention to be packaged into nanoparticles do not requiredelivery to the cytoplasm. For example, as for other ODN-cationinteractions, ODN release can take place in low-pH compartments such asendosomes.

In another embodiment said nanoparticle is an alginate-PLL nanoparticle.Sodium alginate is a natural polysaccharide with mannuronic andguluronic acid as its constituents. Aynié et al. (Antisense Nucleic AcidDrug Dev. 1999; 9:301-12) have described alginate nanoparticlescross-linked by PLL to which ODN are bound. The preparation of theparticles is a two-step process, where an initial alginate pre-gel isproduced by the addition of calcium chloride under magnetic stirring,and second PLL is added to form a polyelectrolyte complex with theremaining free charges of the pre-gel. ISS-NA is added eithersimultaneously with PLL to the pre-gel, or after nanoparticle formation.In a preferred embodiment said nanoparticle comprises or alternativelyessentially consists of Polylysine polymers of MW 3900 and 7900, whereinpreferably said ISS-NA is an unethylated CpG-containing oligonucleotide.Aynié et al. report particle sizes of 200-600 nm, and ODN loading ofapproximately 10%. The loading capacity of the alginate nanoparticleshowever did not reach saturation under the conditions described by Ayniéet al., while the encapsulation efficiency was 100%. Alginate-PLLnanoparticles have thus a high ODN loading capacity and are used inembodiments of the invention to package ODN or ISS-NA.

In a further preferred embodiment, the nanoparticle is a chitosannanoparticle. Chitosan is a positively charged polysaccharide polymerprepared from chitin by deacetylation. The preparation of chitosannanoparticles has been described for example by Roy K. et al. (Nat. Med.1999; 5:387-91) or Mao H Q et al. (J Control Release. 2001; 70:399-421),who obtained nanoparticles of 150-300 nm in size. These authors used acoacervation process, whereby a Chitosan solution in sodium acetate, ismixed to a solution of ISS-NA in sodium sulphate under vortexing at 55°C. Parameters such as temperature, pH, concentration of chitosan, ofsodium sulphate, molecular weight of chitosan and DNA may be varied toobtain optimal nanoparticles. In some embodiments the chitosannanoparticle are further reacted with an N-hydroxy-succinimidederivative of PEG (Leong K W. et al. J Control Release. 1998;53:183-193). In other embodiments the chitosan nanoparticles are furtherconcomitantly reacted with the homobifunctional cross-linking agent DSS(a bis-N-hydroxysuccinimide compound) (Leong K W. et al. J ControlRelease. 1998; 53:183-193). In one embodiment, the ISS-NA is anunmethylated CpG-containing oligonucleotide.

In a further preferred embodiment, said nanoparticle is acationized-gelatin nanoparticle, wherein preferably said ISS-NA is anunmethylated CpG-containing oligonucleotide, preferably G10 (SEQ IDNO:27). The preparation of cationized-gelatin nanoparticle has beendescribed by Zwiorek K. et al. (J Pharm Pharmaceut Sci 2004; 7:22-28) orZillies J and Coester C (J Pharm Pharmaceut Sci 2004; 7:17-21), whoreport nanoparticles with sizes of 180-280 nm. A two-step desolvationtechnique is used to generate nanoparticles, which after resuspension ina buffer at pH 4.5, are further derivatized with cholamine in thepresence of EDC. ISS-NA is subsequently packaged in the nanoparticles byresuspending the nanoparticles in a solution of ISS-NA and furtherincubating the resulting mixture. Zwiorek et al. obtained particles ofsizes ranging from 180-290 nm. Further guidance in preparing gelatinnanoparticles is provided by Azarmi S. et al. (J. Pharm. Pharmaceut.Sci. 2006; 9:124-132), who uses glutaraldehyde instead of EDC forcross-linking the nanoparticles.

In a further preferred embodiment said nanoparticle is a gelatinnanosphere, wherein preferably said ISS-NA is an unmethylatedCpG-containing oligonucleotide. Gelatine nanospheres are prepared asdescribed by Truong-Le V L et al. Hum Gene Ther.1998; 9:1709-1717), by acoacervation technique with Na₂SO₄ at low pH, and whereby the particlesare cross-linked with EDC but omitting transferrin in the reactionmixture. Particle of sizes 300-600 nm were obtained by Truong-Le et al.In a preferred embodiment said nanoparticle is a gelatine-cholaminnanoparticle.

In a further preferred embodiment, said nanoaparticle is an albuminnanoparticle, wherein preferably said ISS-NA is an unmethylatedCpG-containing oligonucleotide. Preparation of albumin nanoparticles hasbeen described, for example, by Irache J M et al. (Mini Rev Med. Chem.2005; 5:293-305), who report nanoparticles with a size of 250-300 nm. Invery preferred embodiment, the albumin nanoparticles are made from humanserum albumin. Albumin nanoparticles are prepared by a coacervation ordesolvation process, and subsequently stabilized by cross-linking withglutaraldehyde. In one method, ISS-NA is incubated with an albuminaqueous solution (2% w/v), and the mixture is desolvated with ethanol,which induces formation of nanoparticles. These are then cross-linkedwith glutaraldehyde. As described by Wartlick et al. (J Control Release.2004; 96:483-495), stability of the nanoparticles can be optimized byadjusting the concentration of glutaraldehyde during the cross-linkingstep. In one further embodiment, the albumin nanoparticle is aprotamine-albumin-ISS-NA nanoparticle (Vogel V. et al. J controlrelease. 2005; 103: 99-11).

In one further embodiment, the albumin nanoparticle is aprotamine-albumin-ISS-NA nanoparticle (Weyermann et al. J controlrelease. 2004; 100: 411-423; Vogel V. et al. 3 control release. 2005;103: 99-11; Mayer G. et al. J control release 2005; 106:181-187).Nanoparticles useful in the practice of the invention also include thenanoparticles described by Kumar MNVR (J. Pharm. Pharmaceut. Sci. 2000;3:234-258).

In a further preferred embodiment, said nanoparticle is amethacrylate-based hydrogel nanoparticle. Preparation of suchnanoparticle as well as the adsorption of ISS-NA has been described byJain S. et al. (Biomacromolecules. 2005; 6:2590-600), who obtainedparticles of about 500 nm in size. These nanoparticles are prepared by atwo-phase miniemulsion polymerization process. The emulsion is formed ina near saturated salt solution of pluronic F-68 as described by Jain S.et al, except that no ovalbumin is included in the process.PEG-methacrylate, methacrylic acid and PEG-dimethacrylate are added tothe pluronic salt solution under stirring. The resulting solution isheated to 40° C., causing phase separation and emulsion, andpolymerization is initiated by addition of ammonium persulfate andsodium meta bisulfite. The nanoparticles are isolated by centrifugation,and poly(L-arginine) followed by ISS-NA are adsorbed onto thehydrophobic nanoparticles.

In a further preferred embodiment, said nanoparticle is a methylmethacrylate-based cationic nanoparticle. Preparation of suchnanoparticles has been described by Tondelli L. et al. (J Biomater SciPolymer Edn 2003; 14:1209-1227), who obtained nanoparticles of 500-1000nm. Methylmethacrylate is mixed with a PEG-derivatized methylmethacrylate and a methacryl methacrylate derivative containing aquaternary ammonium ion in an emulsion polymerization reaction initiatedwith potassium persulfate. ISS-NA is then incubated with the cationicnanoparticle.

In a further preferred embodiment, said nanoparticle is a PLL-modifiedsilica nanoparticle. Preparation of the nanoparticles is described byZhu S G. et al. (Biotechnol Appl Biochem 2004; 39:179-187), who obtainednanoparticles of 20 nm in size. The nanoparticles are prepared using awater-in-oil microemulsion where hydrolysis and condensation reactionsof tetraethoxysilane is initiated with ammoniumhydroxide. Silicananoparticles are activated in carbonate buffer before addition of PLL,and ISS-NA is further incubated with washed PLL-silica nanoparticles.

Nanoparticles suitable in the practice of the invention can be testedfor activation of bone marrow derived dendritic cells (BMDCs) asdescribed in Example 11. Alternatively, in addition to interleukin-12(IL-12) secretion, activity of the nanoparticle in this assay may beidentified by detecting interleukin-6 (IL-6) or IFN-alpha insupernatants, or by quantifying CD-86 upregulation on BMDCs uponnanoparticle incubation by fluorescent associated cell-sorting.Importantly, these assays are reproducible when using an identical batchof BMDCs, which can be frozen. Comparison between active agents shouldtherefore be performed with the same batch of BMDCs.

In a preferred embodiment, nanoparticles with unmethylatedCpG-containing oligonucleotide induce activation of BMDCs in thecellular assay described in Example 11 in a similar way to Qβ packagedwith G10 oligonucleotide and reach the lymph node upon subcutaneousinjection in mice footpad with a kinetic similar to Qβ-VLP or apolystyrene bead of 20-500 nm size (see Examples 7 and 9). Activation ofBMDCs in a way similar to Qβ packaged with G10 oligonucleotide is meantto express that an identical dose of G10 oligonucleotide package innanoparticles give a half-maximal amount of IL-12 secretion within 80%,preferably 60%, more preferably 50%, 40% and 30% of the amount inducedby the same dose of G10 oligonucleotide packaged in Qβ. In a furtherpreferred embodiment, the nanoparticles are protective in the animalmodels of allergy described in the Examples.

In a preferred embodiment the nanoparticle of the invention is packagedwith ISS-NA, preferably with unmethylated CpG-containingoligonucleotide, most preferably with QβG10, amounting to 0.5 to 80%(w/w) of said particle, preferably 0.5 to 40% (w/w), more preferably 2to 40% (w/w), still more preferably 6 to 40% (w/w), even more preferably10 to 40% (w/w), even more preferably 15 to 40% (w/w), most preferably18 to 30% (w/w).

Processes for the preparation of nanoparticles have been reviewed inMicroparticulate systems for the delivery of proteins and vaccines, Eds.S. Cohen and H. Bernstein, Marcel Dekker, New York 1996). Themicroparticles described therein include polyester microparticles,pegylated-polyester microparticles, polyphosphazene microparticles,lipospheres and gelatin microparticles. Additional microparticlesinclude alginate microparticles (Aggarwal N. et al. Can J Vet Res. 1999;63:148-52), chitosan microparticles (Aral C and Akbuga J. J Pharm PharmSci. 2003; 6:321-6; Xu W. Et al. Vaccine. 2004; 22:3603-12). Processesused for the preparation of nanoparticles where resulting particle sizeis determined by the size of the droplet within the emulsion used toprepare the nanoparticle, can be readily adapted for the preparation ofmicroparnicles, as is well-known in the art, whereby the mixing,vortexing, high-speed homogenization steps are modified such thatmicroparticles are produced. The spra-drying method may also be adaptedsuch that microparticles are generated. Microparticles useful in thepractice of the invention also include the microparticles described inKumar MNVR (J Pharm Pharmaceut Sci 2000; 3:234-258).

Processes for packaging of ISS-NA into nanoparticles described above canbe adapted for packaging ISS-NA into microparticles, whereby thehomogenization or mixing step is modified as would be known to thoseskilled in the art, such that the emulsion produced result inmicroparticle generation. The spray-drying method may also be adaptedsuch that microparticles are generated.

The properties of polyester microsphere, and PLGA microsphere inparticular, as well as methods to produce them have been reviewed anddescribed for example by Kissel T and Koneberg R (in Microparticulatesystems for the delivery of proteins and vaccines, Eds. S. Cohen and H.Bernstein, Marcel Dekker, New York 1996, p. 51-87). These methodsinclude spray drying methods, water-in-oil-in-water emulsion solventevaporation methods and phase separation methods. A favored molar ratioof the monomers constituting PLGA is 50% mol LA and 50% mol GA.Increasing the proportion of either of the monomer leads to slowerdegradation of the polymer. For example, a polymer with a ratio of LA toGA of 85:15 has a rate of degradation about two- and a half time slowerthan with a ratio of 50:50. Thus, the porperties of PLGA polymer can bemanipulated by changing the proportion of the monomers.

In one embodiment said synthetic particle is a liposome, wherein saidliposome is a lipid vesicles consisting of a lipid bilayer. Liposomescan be packaged with ISS-NA using methods known in the art. The liposomeof the invention may be selected from the group consisting of neutralliposome, anionic liposome, cationic liposome, stealth, or cationicstealth. In a preferred embodiment, the liposome is a cationic liposome.The liposome may have a diameter between 100 and 800 nm, preferablybetween 100 and 400 nm, more preferably between 100 and 300 nm, evenmore preferably between 100 and 200 nm, most preferably 200 nm.

In a preferred embodiment, the liposome exhibits positive charges inorder to facilitate interaction of liposomes with target cells. In someembodiments, the liposome comprises a cationic lipid, a colipid, and astabilizing additive. In another embodiment, the liposome comprisesdimethylaminoethane-carbamol-cholisterol, and/ordioleoylphosphatidylethanolamine, and/or polyethylene glycol derivatizedphosphatidylethanolamine. In a preferred embodiment, the liposomecomprises phosphatidylcholine, and/or cholesterol, and/orDL-α-tocopherol, preferably phosphatidylcholine, cholesterol, andDL-α-tocopherol. Generation of such liposomes is well established e.g.in Bangham et al., (1965), J. Mol. Biol., 13, 238-252; Gursel et al.,(2001), J Immunol 167: 3324; or Ludewig et al., (2000), Vaccine, 19,23-32, the disclosure of which is incorporated herein by reference inits entirety.

Preferred liposomes and the packaging of ISS-NA in liposomes isdescribed in WO2005/014110A1, which is incorporated herein by reference.ISS-NA may be mixed with preformed vesicles comprising or preferablyessentially consisting of, most preferably consisting of cationiclipids, may be mixed directly with cationic lipid, resulting inlipoplexes, or in a preferred embodiment, encapsulated within theaqueous space enclosed by a lipid bilayer. In a further embodiment, saidliposome is a lipopolyplex (Pelisek J. et al. J Gene Med 2006;8:186-197). In one embodiment, the liposome is a microencapsulatedliposome, in an alginate-PLL coat, as described by Cohen S. et al. Proc.Natl. Acad. Sci. USA 1991; 88:10440-10444). In a preferred embodiment,the ISS-NA, preferably an unmethylated CpG-containing oligonucleotide,is packaged within a “stabilized antisense-lipid particle” containingpreferably PEG-ceramide-C14, as described by Semple S. C. et al. MethodsEnzymol. 2000; 313:322-41. In the performance of this method for thepractice of the invention, the antisense oligonucleotide is replaced byan ISS-NA, and in particular an unmethylated CpG-containingoligonucleotide. These liposomes are prepared with cationic lipids thatare only charged at subphysiological pH. Hence the ISS-NA orunmethylated CpG-containing oligonucleotide bound to the outer surfaceof liposomes during the liposome preparation at low pH can besubsequently dissociated and eliminated by anion exchange chromatographyonce the preparation has been brought back to neutral pH. Suitablefurther liposomes, methods of preparation as well as methods for ISS-NAand in particular oligonucleotides, can be found in Semple S. C. et al.Methods Enzymol. 2000; 313:322-41 and references therein. Methods forpreparing liposomes include the dry lipid hydration method, the reversephase hydration method, the detergent dialysis method, the minimalvolume entrapment method. In certain embodiments, packaging of theISS-NA in particular an unmethylated CpG-containing oligonucleotide isfacilitated by using an ISS-NA or unmethylated CpG-containingoligonucleotide substituted by a residue selected from the groupconsisting of C6-C30 alkyl chain, bile acids, cholic acid, taurocholicacid, desoxycholate, cholesterol, oleyl litocholic acid, oleoyl cholenicacid, glycolipids, phospholipids, sphingolipids, isoprenoids, steroids,vitamins, vitamin E, saturated fatty acids, unsaturated fatty acids,fatty acid esters, triglycerides.

In one aspect of the invention, the ISS-NA in liposomes are used toinduce systemically increased levels of IFN-alpha. Such elevated levelsof IFN-alpha are known to be therapeutically active in hypersensitivity,preferably allergy.

In a further embodiment, said synthetic particle is a virosome, whereinpreferably said visosome is a reconstituted virus envelope of ainfluenza virus, wherein further preferably said influenza virus is ainfluenza A virus, wherein still further preferably said influenza Avirus is influenza A/Singapore virus. Virosomes comprising cationic(positively charged) lipids are especially suited to deliver nucleicacids to a target cell. In a further embodiment, said synthetic particleis a virosome, wherein said virosome comprises a lipid membrane, whereinsaid lipid membrane comprises or preferably essentially consists ofcationic lipids. In a very preferred embodiment, said synthetic particleis a virosome and said ISS-NA is a unmethylated CpG-containingoligonucleotide, wherein preferably said unmethylated CpG containingoligonucleotide is G10 (SEQ ID NO:27), and wherein further preferablysaid virosome comprises a lipid membrane, wherein said lipid membranecomprises or preferably essentially consists of cationic lipids. In afurther preferred embodiment said virosome comprises a lipid membrane,wherein said lipid membrane comprises antibodies or fragments thereof,wherein preferably said antibodies specifically interact with a receptorof a target cell.

Synthetic particles of the invention, preferably microparticles andnanoparticles, most preferably nanoparticles may be injectedsubcutaneously, intravenously, intradermally, intraperitoneally,administered intranasally, orally, transdermally or inhaled.

In a very preferred embodiment said particle is a virus particle or avirus-like particle (VLP), preferably a VLP. Any virus known in the artmay be selected as a VLP or a virus particle of the invention. Mostcommonly known viruses have been sequenced and are readily available tothe public. The taxonomy of viruses is well known to the artisan andsummarized, for example, in H. V. Van Regenmortel et al. (eds.), VirusTaxonomy: 7^(th) Report of the International Committee on Taxonomy ofViruses (2000) (Academic Press/elsevier, Burlington Mass., USA), on theVirus Taxonomy web-page of the University of Leicester (UK)(http://www-micro.msb.le.ac.uk/3035/Virusgroups.html) and by theTaxonomy Browser of the National Center for Biotechnology Information(NCBI, Washington D.C., USA) (http://www.ncbi.nlm.nih.gov/ICTVdb/). Thegenes encoding viral coat proteins can be identified by a skilledartisan and their nucleotide and amino acid sequences may, for example,be obtained from Genbank (http://www.ncbi.nlm.nih.gov/). Viruses whichare particularly useful in the context of the invention are generallydisclosed in “Artificial DNA—Methods and Applications”, Yury Khudyakovand Howard Fields, eds., CCR Press, 2003.

Virus particles or VLPs can be produced and purified from virus-infectedcell cultures. For the purpose of the invention, said virus particles orVLPs are be preferably non-replicative or non-infectious, morepreferably non-replicative and non-infectious. UV irradiation, chemicaltreatment, such as with formaldehyde, β-propione or chloroform, are thegeneral methods known to skilled person to inactivate a virus.Alternatively, said non-replicative and non-infectious virus particle orsaid non-replicative and non-infectious VLP can be produced bypurification and reassembly of core proteins of said virus.

In one embodiment said virus particle or VLP, preferably VLP, is a virusparticle or VLP, preferably VLP, of a virus, wherein said virus may be aDNA virus, including DNA reverse transcribing viruses, or a RNA virus.In a preferred embodiment said virus is a DNA virus, wherein said DNAvirus is a single stranded DNA virus, wherein said single stranded DNAvirus is preferably selected from the group consisting of: (a)Parvovirus, preferably parvovirus B19, porcine parvovirus (PPV) orcanine parvovirus (CPV), (b) Erythrovirus, (c) Dependovirus, (d)recombinant of CPV with feline panleucopenia virus (FPV) (Saliki. T. J.et al. (1992) J. Gen. Virol 73:369ff), (e) adeno-associated virus type 2(AAV-2), (f) mink enteritis parvovirus (MEV), (g) muscovy duckparvovirus (DPV), (h) minute virus of mice (MVM), (i) aleutian minkdisease parvovirus (ADV), and (j) Galleria mellonella densovirus(GMDNV).

In a further preferred embodiment said DNA virus is a double strandedDNA virus, including double stranded DNA reverse transcribing viruses,wherein said double stranded DNA virus is preferably selected from thegroup consisting of: (a) nucleopolyhedrovirus, preferably Autograpacalifornica nucleopolyhedrovirus (AcMNPV) or a chimera of AcMNPVpolyhedrin and Trichoplusioa ni granulosis virus (TnGV) (Eason J. E. etal. (1998), J Virol 72:6237ff), (b) papillomavirus, preferably selectedfrom (i) human papilloma virus (HPV, most preferably HPV6, HPV11, HPV16,HPV18, or HPV33), (ii) bovine papillomavirus (BPV, preferably BPV1), and(iii) cottontail rabbit papillomavirus (CRPV), (c) polyomavirus,preferably selected from (i) murine polyomavirus (preferably Py orSV40), (ii) budgerigar fledgling virus, (iii) human polyomavirus JC,(iv) hamster polyomavirus (HaPV), (v) monkey B-lympotropic papovirus(LPV), (vi) avian polyomavirus (APV) and (vii) recombinant human andnon-human polyomaviruses (Sasnauskas K. et al (1999) Biol. Chem. 380,381), (d) spleen necrosis virus (SNV, Jiang A. (1999) Hum. Gene Therapy10(16):2627-2636), and, very preferably, (e) Hepatitis B virus.

In a further preferred embodiment said virus is a RNA virus, whereinsaid RNA virus may be a single stranded RNA virus or a double strandedRNA virus. In a further preferred embodiment said RNA virus is a singlestranded RNA virus, wherein preferably said single stranded RNA virus isa single stranded positive sense RNA virus, wherein preferably saidsingle stranded positive sense RNA virus is selected from: (a)bromoviridae, preferably selected from (i) alfamovirus (e.g. alfalfamosaic virus (AIMV)), and (ii) ilarvirus (e.g. prunus necrotic ringspotilarvirus (PNRSV, Pallas V. (1998) Arch. Virol. 144:797-803); prunedwarf virus (PDV, Abou-Jawdah Y. et al. (2004) J. Virological Melhods121:31-38)), (iii) bromovirus (e.g. cowpca chlorotic mottle virus (CCMV)or brome mosaic virus (BMV)), (iv) cucumovirus (e.g. cucumber mosaicvirus, Natilla A. et al. Arch Virol 2004 149(1):137-154), (b)tombusviridae, preferably (i) tombusvirus, preferably tomato bushy stuntvirus (TBSV, Joelson T. et al. (1997) J. Gen. Virol. 78:1213-1217), (ii)carmovirus, turnic crinkle virus (TCV, Qu F. and Morris T. J. (1997) J.Virol. 71(2):1428-1435), (c) potyvirus, preferably Johnsongrass mosaicvirus (JGMV) and plum pox potyvirus (PPV, Fernandez-Fernandes M. R. etal. (2002) J. Virol. 76(24):12646-12653), (d) tobacco mosaic virus(TMV), (e) comovirus, preferably cowpea mosaic virus (CPMV), (f) potatovirus X (PVX, Marusic C. et al. (2001) J. Virol. 75(18):8434-8439, (g)calicivirus, preferably selected from (i) norwalk virus (NV), (ii)norwalk-like calcivirus, (iii) human calcivirus, (iv) Lorsdalecalcivirus, (v) rabbit hemorrhagic disease virus (RHDV), (vi) Europeanbrown hare syndrom virus (EBHSV), (vii) Toronto virus, (viii) Hawaiivirus, (ix) Sapporo-like virus, and (x) Grimsby feline calcivirus, (h)RNA bacteriophage, (i) luteovirus, preferably potato leaf roll virus(PLRV), (j) flock house virus, (k) retroid viruses, preferably selectedfrom (i) oncoretrovirus, (ii) lentivirus, and (iii) yeastretrotransposon Tyl, (l) tick-borne encephalitis virus (TBEV, Leibl H.(1998) Vaccine 16(4):340-345) and (m) togaviridae, preferablyalphavirus, most preferably Sindbis virus.

In a further preferred embodiment said RNA virus is a single strandedpositive sense RNA virus selected from: (a) bromoviridae, preferablyselected from (i) alfamovirus (e.g. alfalfa mosaic virus (AIMV)), and(ii) ilarvirus (e.g. prunus necrotic ringspot ilarvirus (PNRSV, PallasV. (1998) Arch. Virol. 144:797-803); prune dwarf virus (PDV, Abou-JawdahY. et al. (2004) J. Virological Methods 121:31-38)), (iii) bromovirus(e.g. cowpea chlorotic mottle virus (CCMV) or brome mosaic virus (BMV)),(iv) cucumovirus (e.g. cucumber mosaic virus, Natilla A. et al. ArchVirol 2004 149(1):137-154), (b) tombusviridae, preferably (i)tombusvirus, preferably tomato bushy stunt virus (TBSV, Joelson T. etal. (1997) J. Gen. Virol. 78:1213-1217), (ii) carmovirus, turnic crinklevirus (TCV, Qu F. and Morris T. J. (1997) J. Virol. 71(2):1428-1435),(c) potyvirus, preferably Johnsongrass mosaic virus (JGMV) and plum poxpotyvirus (PPV, Fernandez-Fernandes M. R. et al. (2002) J. Virol.76(24):12646-12653), (d) tobacco mosaic virus (TMV), (e) comovirus,preferably cowpea mosaic virus (CPMV), (f) potato virus X (PVX, MarusicC. et al. (2001) J. Virol. 75(18):8434-8439, (g) calicivirus, preferablyselected from (i) norwalk virus (NV), (ii) norwalk-like calcivirus,(iii) human calcivirus, (iv) Lorsdale calcivirus, (v) rabbit hemorrhagicdisease virus (RHDV), (vi) European brown hare syndrom virus (EBHSV),(vii) Toronto virus, (viii) Hawaii virus, (ix) Sapporo-like virus, and(x) Grimsby feline calcivirus, (h) RNA bacteriophage, (i) luteovirus.preferably potato leaf roll virus (PLRV), (j) flock house virus, (k)retroid viruses, preferably selected from (i) oncoretrovirus, (ii)lentivirus, and (iii) yeast retrotransposon Tyl, (l) tick-borneencephalitis virus (TBEV, Leibl H. (1998) Vaccine 16(4):340-345), (m)togaviridae, preferably alphavitus, most preferably Sindbis virus, and(n) Nodaviridae, preferably Alphanodavirus, most preferably Pariacotovirus (Johnson K. N. et al. (2004) Journal of Virology 78:11371-11378).

In a further preferred embodiment said RNA virus is a double strandedRNA virus, wherein preferably said double stranded RNA virus is selectedfrom: (a) birnavirus, (b) cypovirus, preferably Bombyx mori cytoplasmicpolyhedrovirus (BmCPV), (c) orbivirus, preferably bluetoung virus (BTV)or African horse sickness virus (AHSV), (d) rotavirus and, verypreferably, (e) double stranded RNA bacteriophages, preferably selectedfrom (i) bacteriophage 8, (ii) bacteriophage phi6, (iii) bacteriophagephi12, and (iv) bacteriophage phi12.

In a further preferred embodiment said virus particle or VLP is a virusparticle or VLP of a virus, wherein said virus is a bacteriophage,wherein said bacteriophage may be a DNA bacteriophage or an RNAbacteriophage.

In a preferred embodiment said bacteriophage is a DNA bacteriophage,wherein said DNA bacteriophage may be a single stranded DNAbacteriophage or a double stranded bacteriophage. In a preferredembodiment, said DNA bacteriophage is a single stranded DNAbacteriophage, wherein said single stranded DNA bacteriophage ispreferably selected from (a) Microviridae, preferably Phi X 174 and (b)Inoviridae, preferably fd and M13. In a further preferred embodiment,said DNA bacteriophage is a double stranded DNA bacteriophage, whereinsaid double stranded DNA bacteriophage is preferably selected from thegroup consisting of: (a) Myoviridae, preferably T2, T4 or T6, (b)Siphoviridae, preferably bacteriophage Lambda, T1, T5 or HK97, (c)Podoviridae, preferably T2, T7 or P22, (d) Tectiviridae, preferablyPRD1, (e) Corticoviridae, preferably PM2, (f) Plasmaviridae, preferablymycoplasma phages, (g) Lipothrixviridae, preferably Thermoproteusbacteriophage TTV1 and (h) Fuselloviridae, preferably sulfolobusbacteriophage 1.

In a more preferred embodiment said bacteriophage is an RNAbacteriophage, wherein said RNA bacteriophage may be a single strandedor a double stranded RNA bacteriophage. In one embodiment said RNAbacteriophage is a single stranded RNA bacteriophage, wherein preferablysaid single stranded RNA bacteriophage is an enterobacteriophage,wherein preferably said enterobacteriophage is a representative of theLeviviridae, wherein preferably said representative of the Leviviridaeis selected from the group consisting of: (a) taxonomically not assignedfamily member Acinetobacter phage 205 (AP205), (b) levivirus, and,preferably (c) allolevivirus.

In a preferred embodiment said representative of the Leviviridae is alevivirus, wherein preferably said levivirus is selected from the groupconsisting of: (a) bacteriophage BZ13, (b) bacteriophage GA, (c)bacteriophage JP34, (d) bacteriophage KU1, (d) bacteriophage TH1, (e)bacteriophage MS2, (f) bacteriophage f2. (g) bacteriophage fr, (h)bacteriophage JP501, (i) bacteriophage M12, (j) bacteriophage R17, and(k) bacteriophagc PP7.

In a more preferred embodiment said representative of the Leviviridae isan allolevivirus, wherein preferably said allolevivirus is selected fromthe group consisting of: (a) bacteriophage FI, (b) bacteriophage ID2,(c) bacteriophage NL95, (d) bacteriophage SP, (d) bacteriophage TW28,(e) bacteriophage Qβ, (f) bacteriophage M11, (g) bacteriophage MX1, (h)bacteriophage ST, (i) bacteriophage TW18, and (j) bacteriophage VK.

In a further preferred embodiment said RNA bacteriophage is selectedfrom the group consisting of: (a) bacteriophage BZ13, (b) bacteriophageGA, (c) bacteriophage JP34, (d) bacteriophage KU1, (d) bacteriophageTH1, (e) bacteriophage MS2, (f) bacteriophage f2, (g) bacteriophage fr,(h) bacteriophage JP501, (i) bacteriophage M12, (j) bacteriophage R17,(k) bacteriophage PP7, (l) bacteriophage FI, (m) bacteriophage ID2, (n)bacteriophage NL95, (o) bacteriophage SP, (p) bacteriophage TW28, (q)bacteriophage Qβ, (r) bacteriophage M11, (s) bacteriophage MX1, (t)bacteriophage ST, (u) bacteriophage TW18, and (v) bacteriophage VK. In afurther preferred embodiment said RNA bacteriophage is selected from thegroup consisting of: (a) bacteriophage Qβ, (b) bacteriophage R17, (c)bacteriophage fr, (d) bacteriophage GA, (e) bacteriophage SP, (f)bacteriophage MS2, (g) bacteriophage M11, (h) bacteriophage MX1, (i)bacteriophage NL95, (k) bacteriophage f2, (l) bacteriophage PP7, and (m)bacteriophage AP205.

In a further preferred embodiment said RNA bacteriophage is a doublestranded RNA bacteriophage, wherein preferably said double stranded RNAbacteriophage is a representative of the Cystoviridae, more preferablysaid representative of the Cystoviridae is a Cystovirus, most preferablysaid Cystovirus is pseudomonas bacteriophage Phi 6.

In a preferred embodiment said particle is a virus particle of abacteriophage, and wherein preferably said bacteriophage is a RNAbacteriophage, wherein further preferably said RNA bacteriophage is asingle stranded positive sensr RNA bacteriophage. and wherein stillfurther preferably said single stranded positive sense RNA bacteriophageis a single stranded positive sense RNA bacteriophage selected from thegroup consisting of: (a) bacteriophage Qβ, (b) bacteriophage fr, (c)bacteriophage GA, and (d) bacteriophage AP205, most preferably saidsingle stranded positive sense RNA bacteriophage is Qβ.

In a very preferred embodiment, said particle is a VLP, preferably a VLPof an RNA virus, more preferably a VLP of a single stranded positivesense RNA virus, most preferably a VLP of an RNA bacteriophage.

In a further preferred embodiment said particle is a VLP of abacteriophage, more preferably a VLP of a enterobacteriophage, stillmore preferably a VLP of a representative of the Leviviridae, mostpreferably a VLP of a levivirus or an allolevivirus. I a very preferredembodiment said VLP is a VLP of an allolevivirus.

In a further embodiment said particle is a virus particle or a VLP,preferably a VLP, of a icosahedral virus, wherein said icosahedral virusis preferably a plant-infectious icosahedral virus. VLPs ofplant-infectious icosahedral viruses are for example disclosed inWO2005/067478A2 which is incorporated herein by reference. In apreferred embodiment said icosahedral virus is selected from arepresentative of any one taxon selected from the group consisting of(a) Papillomaviridae, (b) Totiviridae, (c) Dcistroviridae, (d)Hepadnaviridae, (e) Togaviridae, (f) Polyomaviridae, (g) Nodaviridae,(h) Tectiviridae, (i) Leviviridae, (j) Microviridae, (k) Sipoviridae,(l) Picornoviridae, (m) Parvoviridae, (n) Calciviridae, (O)Tetraviridae, and (p) Satellite viruses. In a preferred embodiment, saidicosahedral virus is a plant-infectious icosahedral virus, wherein saidplant-infectious icosahedral virus is a representative of any one taxonselected from the group consisting of (a) Bunyaviridae, (b) Reoviridae,(c) Rhabdoviridae, (d) Luteoviridae, (e) Nanoviridae, (f)Partitiviridae, (g) Sequiviridae, (h) Tymoviridae, (i) Ourmiavirus, (j)Tobacco Necrosis Virus Satellite, (k) Caulimoviridae, (l) Geminiviridae,(m) Comoviridae, (n) Sobemovirus, (O) Tombusviridae, and (p)Bromoviridae. In a further preferred embodiment, said plant-infectiousicosahedral virus is a representative of any one taxon selected from thegroup consisting of (a) Luteoviridae, (b) Nanoviridae, (c)Partitiviridae, (d) Sequiviridae, (e) Tymoviridae, (f) Ourmiavirus, (g)Tobacco Necrosis Virus Satellite, (h) Caulimoviridae, (i) Geminiviridae,(j) Comoviridae, (k) Sobemovirus, (l) Tombusviridae, and (m)Bromoviridae. In a further preferred embodiment, said plant-infectiousicosahedral virus is a representative of any one taxon selected from thegroup consisting of (a) Caulimoviridae, (b) Geminiviridae, (c)Comoviridae, (d) Sobemovirus, (e) Tombusviridae, and (e) Bromoviridae.In a further preferred embodiment, said plant-infectious icosahedralvirus is a representative of any one taxon selected from the groupconsisting of the (a) Comoviridae, (b) Sobemovirus, (c) Tombusviridae.and (d) Bromovirida. In a further preferred embodiment, saidplant-infectious icosahedral virus is a representative of any one taxonselected from Comoviridae and Bromoviridae. In a very preferredembodiment said plant-infectious icosahedral virus is a Cowpea MosaicVirus or a Cowpea Chlorotic Mottle Virus. In a further preferredembodiment said plant-infectious icosahedral virus is a representativeof the Bromoviridae, preferably Bromovirus, Cucumovirus. Ilarvirus orAlfamovirus. In a very preferred embodiment said plant-infectiousicosahedral virus is selected from: brome mosaic virus, cowpea chloroticmottle virus, cucumber mosaic virus, Tobacco streak virus and alfalfamosaic virus (AMV, including AMV1 and AMV2).

In a further preferred embodiment said VLP is a synthetic VLP.

In a further preferred embodiment, the VLP is a recombinant VLP. Thepreparation of VLPs by recombinantly expressing the coat protein in ahost is within the common knowledge of a skilled artisan. IllustrativeDNA or RNA viruses, the coat or capsid protein of which can be used forthe preparation of VLPs have been disclosed in WO 2004/009124 on page25, line 10-21, on page 26, line 11-28, and on page 28, line 4 to page31, line 4. These disclosures are incorporated herein by way ofreference.

In one preferred embodiment, said VLP comprises, or alternativelyconsists of, recombinant proteins, mutants or fragments thereof, of avirus, wherein preferably said virus is selected from any virus listedabove. In a very preferred embodiment said VLP comprises, oralternatively consists of, recombinant proteins, mutants or fragmentsthereof, of a virus, wherein said virus is selected form the groupconsisting of: (a) RNA bacteriophages, (b) bacteriophages, (c) HepatitisB virus, preferably its capsid protein (Ulrich, et al., Virus Res.50:141-182 (1998)) or its surface protein (WO 92/11291), (d) measlesvirus (Warnes, et al., Gene 160:173-178 (1995)), (e) Sindbis virus; (f)rotavirus (U.S. Pat. No. 5,071,651 and U.S. Pat. No. 5,374,426), (g)foot-and-mouth-disease virus (Twomey, et al., Vaccine 13:1603 1610,(1995)), (h) Norwalk virus (Jiang, X., et al., Science 250:1580 1583(1990); Matsui, S. M., et al., J. Clin. Invest. 87:1456 1461 (1991)),(i) Alphavirus, (j) retrovirus, preferably its GAG protein (WO96/30523), (k) retrotransposon Ty, preferably the protein p1; (l) humanPapilloma virus (WO 98/15631), (m) Polyoma virus, (n) Tobacco mosaicvirus, and (O) Flock House Virus. In a very preferred embodiment saidVLP comprises, or alternatively consists of, recombinant proteins,mutants or fragments thereof, of a virus, wherein said virus is selectedform the group consisting of: (a) Hepatitis B virus, and (b) Polyomavirus.

In a further preferred embodiment, said VLP comprises, or alternativelyconsists of, recombinant proteins, mutants or fragments thereof, of avirus, wherein said virus is a plant-infectious icosahedral virus,wherein preferably said plant-infectious icosahedral virus is selectedfrom (a) Comoviridae, (b) Sobemovirus, (c) Tombusviridae, and (d)Bromoviridae.

In one preferred embodiment, the VLP comprises or consists of more thanone amino acid sequences, preferably two amino acid sequences, of therecombinant proteins, mutants or fragments thereof. VLP comprises orconsists of more than one amino acid sequence is referred, in thisapplication, as mosaic VLP.

The term “fragment of a recombinant protein” or the term “fragment of acoat protein”, as used herein, is defined as a polypeptide, which is ofat least 70%, preferably at least 80%, more preferably at least 90%,even more preferably at least 95% the length of the wild-typerecombinant protein, or coat protein, respectively and which preferablyretains the capability of forming VLP. Preferably, the fragment isobtained by (i) at least one, preferably exactly one, internal deletion,(ii) at least one, preferably exactly one, truncation, or (iii) at leastone, preferably exactly one, combination thereof. Further preferably,the fragment is obtained by at most 5, 4, 3 or 2 internal deletions, atmost 2 truncations or exactly one combination thereof. Furtherpreferably, the fragment is obtained by at most 5, 4, 3 or 2 internaldeletions, wherein still further preferably each of said deletionscomprises 1 to 5, preferably 1 to 4, more preferably 1 to 3, still morepreferably 1 to 2, and most preferably exactly 1 amino acid.

The term “fragment of a recombinant protein” or “fragment of a coatprotein” shall further encompass polypeptide, which has at least 80%,preferably 90%, even more preferably 95% amino acid sequence identitywith the “fragment of a recombinant protein” or “fragment of a coatprotein”, respectively, as defined above and which is preferably capableof assembling into a virus-like particle.

The term “mutant coat protein” refers to a polypeptide having an aminoacid sequence derived from the wild type recombinant protein, or coatprotein, respectively, wherein the amino acid sequence is at least 80%,preferably at least 85%, 90%, 95%, 97%, or 99% identical to the wildtype sequence and preferably retains the ability to assemble into a VLP.

In one preferred embodiment, the VLP of the invention is VLP ofHepatitis B virus. The preparation of Hepatitis B virus-like particleshas been disclosed, inter alia, in WO00/32227, WO01/85208, WO01/056905and WO2004/000351. All four documents are explicitly incorporated hereinby way of reference. Other variants of HBcAg suitable for use in thepractice of the present invention have been disclosed in page 34-39 ofWO 01/056905. Specifically preferred Hepatitis B virus VLPs aredescribed on page 43, line 12 to page 49, line 8 of WO2004/000351 and inSEQ IDs NO:19-68, 71 and 97 of WO2004/000351. In one further preferredembodiment of the invention, a lysine residue is introduced into theHBcAg polypeptide. In preferred embodiments, VLPs and compositions ofthe invention are prepared using a HBcAg comprising, or alternativelyconsisting of, amino acids 1-144, or 1-149, 1-185 of SEQ ID NO:1, whichis modified so that the amino acids at positions 79 and 80 are replacedwith a peptide having the amino acid sequence of Gly-Gly-Lys-Gly-Gly(SEQ ID NO:24). This modification changes the SEQ ID NO:1 to SEQ IDNO:2. In further preferred embodiments, the cysteine residues atpositions 48 and 110 of SEQ ID NO:2, or its corresponding fragments,preferably 1-144 or 1-149, are mutated to serine. The invention furtherincludes compositions comprising Hepatitis B core protein mutants havingabove noted corresponding amino acid alterations. The invention furtherincludes compositions and pharmaceutical compositions respectively,comprising HBcAg polypeptides which comprise, or alternatively consistof, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97% or99% identical to SEQ ID NO:2.

In one preferred embodiment of the invention, the virus-like particlecomprises, consists essentially of, or alternatively consists of,recombinant coat proteins, mutants or fragments thereof, of a RNAbacteriophage. Preferably, the RNA bacteriophage is selected from thegroup consisting of: (a) bacteriophage BZ13, (b) bacteriophage GA, (c)bacteriophage JP34, (d) bacteriophage KU1, (d) bacteriophage TH1, (e)bacteriophage MS2, (f) bacteriophage f2, (g) bacteriophage fr, (h)bacteriophage JP501, (i) bacteriophage M12, (j) bacteriophage R17, (k)bacteriophage PP7, (l) bacteriophage FI, (m) bacteriophage ID2, (n)bacteriophage NL95, (O) bacteriophage SP, (p) bacteriophage TW28, (q)bacteriophage Qβ, (r) bacteriophage M11, (s) bacteriophage MX1, (t)bacteriophage ST, (u) bacteriophage TW18, and (v) bacteriophage VK.Further preferably, the RNA bacteriophage is selected from the groupconsisting of: (a) bacteriophage Qβ, (b) bacteriophage R17, (c)bacteriophage fr, (d) bacteriophage GA, (e) bacteriophage SP, (f)bacteriophage MS2, (g) bacteriophage M11, (h) bacteriophage MX1, (i)bacteriophage NL95, (k) bacteriophage f2, (l) bacteriophage PP7, and (m)bacteriophage AP205.

In one preferred embodiment of the invention, the virus-like particlecomprises at least one coat protein, mutant or fragment thereof, of anRNA bacteriophage, wherein the coat protein has an amino acid sequenceselected from the group consisting of: (a) SEQ ID NO:3 referring to QβCP; (b) a mixture of SEQ ID NO:3 and SEQ ID NO:4 (Qβ A1 protein); (c)SEQ ID NO:5 (R17 capsid protein); (d) SEQ ID NO:6 (fr capsid protein);(e) SEQ ID NO:7 (GA capsid protein): (f) SEQ ID NO:8 (SP capsidprotein); (g) a mixture of SEQ ID NO:8 and SEQ ID NO:9; (h) SEQ ID NO:10(MS2 capsid protein); (i) SEQ ID NO:11 (M11 capsid protein); (j) SEQ IDNO:12 (MX1 capsid protein); (k) SEQ ID NO:13 (NL95 capsid protein); (l)SEQ ID NO: 14 (f2 capsid protein); (m) SEQ ID NO:15 (PP7 capsidprotein); and (n) SEQ ID NO:21 (AP205 capsid protein). In a furtherpreferred embodiment of the present invention, the virus-like particlecomprises coat proteins having an amino acid sequence selected from thegroup consisting of: (a) SEQ ID NO:3; (b) a mixture of SEQ ID NO:3 andSEQ ID NO:4; (c) SEQ ID NO:6; (d) SEQ ID NO:7; (e) SEQ ID NO:21. In afurther very preferred embodiment of the present invention, thevirus-like particle comprises coat proteins having an amino acidsequence selected from the group consisting of: (a) SEQ ID NO:3; and (b)a mixture of SEQ ID NO:3 and SEQ ID NO:4.

In a further very preferred embodiment of the present invention, thevirus-like particle essentially consists of coat proteins having anamino acid sequence of SEQ ID NO:3, or essentially consists of a mixtureof coat proteins having amino acid sequences of SEQ ID NO: 4, or mutantsthereof, and of SEQ ID NO:3.

In one preferred embodiment of the invention, the VLP is a mosaic VLPcomprising or alternatively consisting of more than one amino acidsequence, preferably two amino acid sequences, of coat proteins, mutantsor fragments thereof, of a RNA bacteriophage.

In one embodiment, the virus particle or VLP is a VLP of bacteriophagefr or GA. Fr coat protein in the form of recombinant VLP may be obtainedas described by Pushko P et al. ((1993) Prot Engin 6:883-891), while GAVLP may be obtained by cloning GA coat protein cDNA isolated by reversetranscription from GA phage into pQb185, which is described for examplein WO2004/007538. Disassembly of Fr and GA VLPs can be readily done byincubating the VLPs in 7 M urea, optionally supplemented with aceticacid at a concentration of 0.1M. The nucleic acid is further purifiedfrom the coat protein by ion exchange chromatography, either at a pHwhere a significant amount of the coat protein flows through while thenucleic acid is retained, or at a pH where the coat protein is alsoadsorbed on the column and subsequently eluted with a salt gradient.Reassembly of fr and GA coat protein with ISS-NA is effected essentiallyas described in WO2003/024481 by slow dialysis, wherein said ISS-NApreferably is an unmethylated CpG-containing oligonucleotide, morepreferably G10 (SEQ ID NO:27), and even more preferably aggregated G10(SEQ ID NO:27) having a retention time relative to Qf capsid standardunder HPLC conditions as set forth in Example 2 of 80 to 120%, mostpreferably of 80 to 95%. At the end of the reassembly reaction thereassembly mixture is concentrated for example by dialysis against a 50%(v/v) glycerol solution in NET buffer (WO2003/024481) and purifiedfurther by gel filtration, for example on a Sepharose CL-4B column.Additional purification methods include ultracentrifugation on a CsClgradient or sucrose cushion. Further protocols for the disassembly andreassembly of Fr and GA VLPs are disclosed in Examples 5 and 6 of thepresent application.

In one very preferred embodiment, the VLP comprises or alternativelyconsists of two different coat proteins of a RNA bacteriophage, said twocoat proteins have an amino acid sequence of CP Qβ (SEQ ID NO: 3) and CPQβ A1 (SEQ ID NO:4), or of CP SP (SEQ ID NO:8) and CP SP A1 (SEQ IDNO:9).

In preferred embodiments of the present invention, the virus-likeparticle of the invention comprises, or alternatively consistsessentially of, or alternatively consists of recombinant coat proteins,mutants or fragments thereof, of the RNA-bacteriophage Qβ, fr, AP205 orGA.

In one preferred embodiment, the VLP of the invention is a VLP of RNAbacteriophage Qβ. The capsid or virus-like particle of Qβshowed anicosahedral phage-like capsid structure with a diameter of 25 nm and T=3quasi symmetry. The capsid contains 180 copies of the coat protein,which are linked in covalent pentamers and hexamers by disulfide bridges(Golmohammadi, R. et al., Structure 4:543-5554 (1996)), leading to aremarkable stability of the Qβ capsid. Capsids or VLPs made fromrecombinant Qβ coat protein may contain, however, subunits not linkedvia disulfide bonds to other subunits within the capsid. or incompletelylinked.

Further preferred VLPs of RNA bacteriophages in accordance with thisinvention, in particular of Qβ and fr, are disclosed in WO 02/056905,the disclosure of which is herewith incorporated by reference in itsentirety. In particular Example 18 of WO 02/056905 gave detaileddescription of preparation of VLP particles from Qβ.

In another preferred embodiment, the VLP of the invention is a VLP ofRNA bacteriophage AP205. Assembly-competent mutant forms of AP205 VLPs,including AP205 coat protein with the substitution of proline at aminoacid 5 to threonine, may also be used in the practice of the inventionand leads to other preferred embodiments of the invention. WO2004/007538 describes, in particular in Example 1 and Example 2, how toobtain VLP comprising AP205 coat proteins, and hereby in particular theexpression and the purification thereto. WO 2004/007538 is incorporatedherein by way of reference. In a further preferred embodiment said virusparticle or VLP is a virus particle or VLP of RNA bacteriophage AP205,wherein said ISS-NA preferably is an unmethylated CpG-containingoligonucleotide, more preferably G10 (SEQ ID NO:27), and even morepreferably aggregated G10 (SEQ ID NO:27) having a retention timerelative to Qβ capsid standard under HPLC conditions as set forth inExample 2 of 80 to 120%, most preferably of 80 to 95%. The disassemblyand reassembly of AP205 is demonstrated in Example 5.

Qβ mutants, of which exposed lysine residues are replaced by argininescan be used for the present invention. Thus, in another preferredembodiment of the present invention, the virus-like particle comprises,consists essentially of or alternatively consists of mutant Qβ coatproteins. Preferably these mutant coat proteins comprise oralternatively consist of an amino acid sequence selected from the groupof a) Qβ-240 (SEQ ID NO: 16, Lys13-Arg of SEQ ID NO: 3) b) Qβ-243 (SEQID NO: 17, Asn10-Lys of SEQ ID NO:3); c) Qβ-250 (SEQ ID NO:18, Lys2-Argof SEQ ID NO:3) d) Qβ-251 (SEQ ID NO:19, Lys16-Arg of SEQ ID NO:3); ande) Qβ-259 (SEQ ID NO:20, Lys2-Arg, Lys16-Arg of SEQ ID NO:3). Theconstruction, expression and purification of the above indicated Qβmutant coat proteins, mutant Qβ coat protein VLPs and capsids,respectively, are described in WO 02/056905. In particular is herebyreferred to Example 18 of above mentioned application.

In a further preferred embodiment said virus particle or VLP is a virusparticle or VLP of RNA bacteriophage Qβ, wherein said ISS-NA preferablyis an unmethylated CpG-containing oligonucleotide, more preferably G10(SEQ ID NO:27), and even more preferably aggregated G10 (SEQ ID NO:27)having a retention time relative to Qβ capsid standard under HPLCconditions as set forth in Example 2 of 80 to 120%, most preferably of80 to 95%. The disassembly and reassembly of Qβ VLPs is demonstrated inExamples 1 and 3.

In another preferred embodiment of the present invention, the virus-likeparticle comprises, or alternatively consists essentially of, oralternatively consists of mutant coat protein of Qβ, or mutants orfragments thereof, and the corresponding A1 protein. In a furtherpreferred embodiment, the virus-like particle comprises, oralternatively consists essentially of, or alternatively consists ofmutant coat protein with amino acid sequence SEQ ID NO:16, 17, 18, 19,or 20 and the corresponding A1 protein.

Further RNA bacteriophage coat proteins have also been shown toself-assemble upon expression in a bacterial host (Kastelein, R A. etal., Gene 23:245-254 (1983), Kozlovskaya, T M. et al., Dokl. Akad. NaukSSSR 287:452-455 (1986), Adhin, M R. et al., Virology 170:238-242(1989), Priano, C. et al., J. Mol. Biol. 249:283-297 (1995)). Inparticular the biological and biochemical properties of GA (Ni, CZ., etal., Protein Sci. 5:2485-2493 (1996), Tars, K et al., J. Mol. Biol.271:759-773 (1997)) and of fr (Pushko P. et al., Prot. Eng. 6:883-891(1993), Liljas, L et al. J. Mol. Biol. 244:279-290, (1994)) have beendisclosed. The crystal structure of several RNA bacteriophages has beendetermined (Golmohammadi, R. et al., Structure 4:543-554 (1996)).

In one preferred embodiment, the virus particle or VLP is a VLP or virusparticle of Cowpea cholortic mottle virus (CCMV). Assembly of CCMV virusfrom coat proteins expressed in E. Coli and nucleic acids has beendescribed (Zhao X. et al. (1995) Virology 207:486-494). In particular,the reassembly of CCMV with RNA was shown to be independent of RNAsequence (Johnson J M. et al. (2004) J Mol Biol 335:455-464).Furthermore, the virus may exist in a swollen form, susceptible tonuclease digestion, which can be disassembled by adding a high NaClconcentration (1M: Johnson J E and Speir J A (1997) J Mol Biol269:665-675). Methods for reassembly of CCMV in the presence of nucleicacids are also described therein. In one embodiment, the CCMV particleis thus reassembled with ISS-NA, preferably with an unmethylatedCpG-containing oligonucleotide. In a very preferred embodiment, theunmethylated CpG-containing oligonucleotide is G10 (SEQ ID NO:27), morepreferably aggregated G10, still more preferably aggregated G10 having aretention time relative to QQ capsid standard under HPLC conditions asset forth in Example 2 of 80 to 120%, most preferably of 80 to 95%. Inone further embodiment, native CCMV virus particle is swollen, treatedwith nucleases, and an ISS-NA, preferably an unmethylated CpG-containingoligonucleotide. and even more preferably G10 (SEQ ID NO:27) is added tothe nuclease treated particle after nuclease removal. In one furtherembodiment, CCMV capsids are reassembled without nucleic acids, as hasbeen described for example by Zlotnick et al. (2000) Virology277:450-456, optionally swollen by bringing the solution to theappropriate pH and ionic strength as described by Zlotnick et al.((2000) Virology 277:450-456) or Johnson J E and Speir J A ((1997) J MolBiol 269:665-675) and ISS-NA, preferably an unmethylated CpG-containingoligonucleotide, and even more preferably G10 (SEQ ID NO:27) is added tothe swollen empty particle.

In one further embodiment, the virus particle or VLP is a VLP or virusparticle of Brome mosaic virus (BMV). Reassembly of BMV has beendescribed previously (Choi Y G and Rao L N (2000) Virology 275: 249-257,and references therein). A tRNA-like structure (tls) at the 3′ of eachviral RNA has been shown to be required for packaging, and can be addedin trans (Choi Y G et al. (2002) Proc. Natl. Acad. Sci. USA 99:655-660)as a nucleotide sequence of about 200 nucleotide in length. T is fromother viruses such as tobacco mosaic virus (TMV) or CMV, or even tRNAssuch as wheat germ tRNAs may also be added in trans and facilitatereassembly, although Choi et al. did not detect packaging of tRNAs inthe BMV capsid (Choi Y G et al. (2002) Proc. Natl. Acad. Sci. USA99:655-660). Thus in one further embodiment, BMV is reassembled with anISS-NA, preferably an unmethylated CpG-containing oligonucleotide, morepreferably G10 and even more preferably aggregated G10 having aretention time relative to Qβ capsid standard under HPLC conditions asset forth in Example 2 of 80 to 120%, most preferably of 80 to 95%.

The compositions of the invention comprise an immunostimulatory nucleicacid (ISS-NA), wherein preferably said ISS-NA is capable of inducing theproduction of a cytokin, preferably of IFN-alpha, in a cell, preferablyin a dendritic cell. In one embodiment, said ISS-NA is selected from thegroup consisting of: (a) ribonucleic acids; (b) desoxyribonucleic acids,(c) chimeric nucleic acids; and (d) any mixtures of at least one nucleicacid of (a), (b) and/or (c). In a preferred embodiment, said ISS-NA is aribonucleic acid, wherein preferably said ribonucleic acid is a doublestranded ribonucleic acid, preferably a double stranded ribonucleic acidselected from the group consisting of: (a) double stranded viral RNA,and (b) synthetic double stranded RNA, preferably poly-(A:U) orpoly(I:C), most preferably poly(I:C).

The innate immune system has the capacity to recognize invariantmolecular pattern shared by microbial pathogens. Recent studies haverevealed that this recognition is a crucial step in inducing effectiveimmune responses. The main mechanism by which microbial products augmentimmune responses is to stimulate APC, especially dendritic cells toproduce proinflammatory cytokines and to express high levelsco-stimulatory molecules for T cells. These activated dendritic cellssubsequently initiate primary T cell responses and dictate the type of Tcell-mediated effector function. Three classes of nucleic acids, namely(i) bacterial DNA that contains immunostimulatory sequences, inparticular unmethylated CpG dinucleotides within specific flanking bases(referred to as CpG motifs), (ii) double-stranded RNA synthesized byvarious types of viruses represent important members of the microbialcomponents that enhance immune responses, and (iii) single stranded RNA.Synthetic double stranded (ds) RNA such as polyinosinic-polycytidylicacid (poly I:C) are capable of inducing dendritic cells to produceproinflammatory cytokines and to express high levels of costimulatorymolecules. A series of studies by Tokunaga and Yamamoto et al. has shownthat bacterial DNA or synthetic oligodesoxynucleotides induce human PBMCand mouse spleen cells to produce type I interferon (IFN) (reviewed inYamamoto et al., Springer Semin Immunopathol. 22:11-19). Poly (I:C) wasoriginally synthesized as a potent inducer of type I IFN but alsoinduces other cytokines such as IL-12. Preferred ribonucleic acidencompass polyinosinic-polycytidylic acid double-stranded RNA (poly LC).Ribonucleic acids and modifications thereof as well as methods for theirproduction have been described by Levy, H. B (Methods Enzymol. 1981,78:242-251), DeClercq, E (Methods Enzymol. 1981, 78: 227-236) andTorrence, P. F. (Methods Enzymol 1981; 78:326-331) and referencestherein. Further preferred ribonucleic acids comprise polynucleotides ofinosinic acid and cytidiylic acid such poly (I:C) of which two strandsform double stranded RNA. Ribonucleic acids can be isolated fromorganisms. Ribonucleic acids also encompass further syntheticribonucleic acids, in particular synthetic poly (I:C) oligonucleotidesthat have been rendered nuclease resistant by modification of thephosphodiester backbone, in particular by phosphorothioatemodifications. In a further embodiment the ribose backbone of poly (I:C)is replaced by a desoxyribose. Those skilled in the art know procedureshow to synthesize synthetic oligonucleotides.

In a further embodiment said ISS-NA is a single stranded ribonucleicacid, preferably polyuridylic acid (poly-U, Westwood A. (2006), Vaccine24:1736-1743). In a preferred embodiment said ISS-NA isdesoxyribonucleic acid, wherein preferably said desoxyribonucleic acidis a double stranded desoxyribonucleic acid. In very preferredembodiment said ISS-NA is desoxyribonucleic acid, wherein preferablysaid desoxyribonucleic acid is a single stranded desoxyribonucleic acid,most preferably a oligodesoxynucleotide (ODN).

In another embodiment, said ISS-NA is an oligonucleotide, wherein saidoligonucleotide is preferably selected from the group consisting of (a)unmethylated CpG-containing oligonucleotide; and (b) oligonucleotidefree of unmethylated CpG motifs. Preferably, said ISS-NA is anunmethylated CpG-containing oligonucleotide. UnmethylatedCpG-dinucleotides within specific flanking bases (referred to as CpGmotifs) represent important members of the microbial components thatenhance immune responses. Toll-like receptor 9 (TLR9) is activated bybacterial DNA, in particular by unmethylated CpG-containingoligonucleotides. In general, the unmethylated CpG-containingoligonucleotide comprises the sequence: 5′ X₁X₂CGX₃X₄ 3′, wherein X₁,X₂, X₃ and X₄ are any nucleotide. Preferred unmethylated CpG-containingoligonucleotides further comprise about 6 to about 100,000 nucleotides,more preferably about 6 to about 2000 nucleotides, still more preferablyabout 20 to about 2000 nucleotides, and even more preferably comprisesabout 20 to about 300 nucleotides. Further preferred unmethylatedCpG-containing oligonucleotides comprise 100 to about 2000 nucleotides,preferably 100 to about 1000 nucleotides, and more preferably 100 toabout 500 nucleotides. Specifically preferred oligonucleotides,unmethylated CpG-containing oligonucleotide, in the context of theinvention comprise 20 to 40, preferably 26, 27, 28, 29, 30, 31 or 32nucleotides, most preferably 30 nucleotides.

The CpG-containing oligonucleotide can contain one or morephosphothioester modifications of the phosphate backbone to enhance thestability of the oligonucleotide. For example, a CpG-containingoligonucleotide having one or more phosphate backbone modifications orhaving all of the phosphate backbone modified and a CpG-containingoligonucleotide wherein one, some or all of the nucleotide phosphatebackbone modifications are phosphorothioate modifications are includedwithin the scope of the present invention. In a preferred embodimentsaid ISS-NA is a CpG-containing oligonucleotide, wherein preferably saidCpG-containing oligonucleotide consisting exclusively of phosphodiesterbound, preferably unmethylated nucleotides are preferred in the contextof the invention.

The CpG-containing oligonucleotide can also be recombinant, genomic,synthetic, cDNA, plasmid-derived and single or double stranded. For usein the invention, the nucleic acids can be synthesized de novo using anyof a number of procedures well known in the art. For example, theb-cyanoethyl phosphoramidite method (Beaucage. S. L., and Caruthers, M.H., Tet. Let. 22:1859 (1981): nucleoside H-phosphonate method (Garegg etal., Tet. Let. 27:4051-4054 (1986); Froehler et al., Nucl. Acid. Res.14:5399-5407 (1986); Garegg et al., Tet. Let. 27:4055-4058 (1986),Gaffney et al. Tet. Let. 29:2619-2622 (1988)). These chemistries can beperformed by a variety of automated oligonucleotide synthesizersavailable in the market. Alternatively, CpGs can be produced on a largescale in plasmids, (see Sambrook, T., et al., “Molecular Cloning: ALaboratory Manual,” Cold Spring Harbor laboratory Press, New York, 1989)which after being administered to a subject are degraded intooligonucleotides. Oligonucleotides can be prepared from existing nucleicacid sequences (e.g., genomic or cDNA) using known techniques, such asthose employing restriction enzymes, exonucleases or endonucleases.

The ISS-NA. preferably the unmethylated CpG-containing oligonucleotide,can be bound to the particle by any way known in the art provided thecomposition enhances an immune response in an animal. For example, theISS-NA can be bound either covalently or non-covalently. Preferably, theparticle, preferably the VLP, encloses, fully or partially, the ISS-NA,preferably the unmethylated CpG-containing oligonucleotide. In a verypreferred embodiment said particle, preferably said VLP, is packagedwith said ISS-NA, wherein further preferably said ISS-NA is aunmethylated CpG-containing oligonucleotide, most preferably G10 (SEQ IDNO:27).

In one embodiment the ISS-NA, preferably the unmethylated CpG-containingoligonucleotide, is bound to the particle, preferably to said VLP, at abinding site, preferably a binding site selected from (a)oligonucleotide binding site (either naturally or non-naturallyoccurring), (b) a DNA binding site, and (c) a RNA binding site. Inanother embodiment, the VLP binding site comprises an arginine-richrepeat or a lysine-rich repeat.

In another preferred embodiment of the present invention, the ISS-NA isan unmethylated CpG-containing oligonucleotide, wherein the CpG motif ofsaid unmethylated CpG-containing oligonucleotide is part of apalindromic sequence, wherein preferably said palindromic sequence isselected from any one of SEQ ID NO:28 and SEQ ID NOs: 35 to 60.Preferably, said palindromic sequence is GACGATCGTC (SEQ ID NO:28).

In a preferred embodiment said ISS-NA is an A-type CpG or an C-type CpG.Preferably, said unmethylated CpG containing oligonucleotide is anA-type CpG, wherein preferably the nucleotides are exclusively linked byphosphodiester bonds. In a further preferred embodiment said ISS-NA is aA-type CpG comprising a palindromic sequence, wherein preferably saidpalindromic sequence is selected from the group consisting of: (a)GACGTC (SEQ ID NO:35), (b) AGCGCT (SEQ ID NO:36), (c) AACGTT (SEQ IDNO:37), (d) ATCGAT (SEQ ID NO:38); (e) CGATCG (SEQ ID NO:39); (f) CGTACG(SEQ ID NO:40); (g) CGCGCG (SEQ ID NO:41); (h) GCGCGC (SEQ ID NO:42);(i) TCGCGA (SEQ ID NO:43); (j) ACGATCGT (SEQ ID NO:44); (k) CGACGATCGTCG(SEQ ID NO:45); (l) CGACGACGATCGTCGTCG (SEQ ID NO:46); (m) GACGATCGTC(SEQ ID NO:28), (n) CGACGACGATCGTCGTCG (SEQ ID NO:47); (O) AACGT (SEQ IDNO:48); (p) CAACGTTG (SEQ ID NO:49); (q) ACAACGTTGT (SEQ ID NO:50); (r)AACAACGTTGTT (SEQ ID NO:51); and (s) CAACAACGTTGTTG (SEQ ID NO:52). In afurther preferred embodiment said ISS-NA is a A-type CpG comprising apalindromic sequence, wherein said palindromic sequence is GACGATCGTC(SEQ ID NO:28).

In a preferred embodiment, said palindromic sequence is flanked at its3′-terminus and at its 5′-terminus by guanosine entities. In a furtherpreferred embodiment said palindromic sequence is flanked at its5′-terminus by at least 3 and at most 25 guanosine entities, whereinsaid palindromic sequence is flanked at its 3′-terminus by at least 3and at most 25 guanosine entities. In a further preferred embodimentsaid palindromic sequence is flanked at its 5′-terminus by at least 3and at most 15, preferably at most 10, guanosine entities, wherein saidpalindromic sequence is flanked at its 3′-terminus by at least 3 and atmost 15, preferably at most 10 guanosine entities. In another preferredembodiment, the palindromic sequence is flanked at its 5′-terminus andat its 3′-terminus by at least 3 and at most 15, preferably at most 10,guanosine entities. In a further preferred embodiment, the palindromicsequence is flanked at its 5′-terminus by at least 3 and at most 15,preferably at most 10, guanosine entities, and wherein said palindromicsequence is flanked at its 3′-terminus by at least 6 and at most 15,preferably at most 10, guanosine entities. In a further preferredembodiment, the palindromic sequence is flanked at its 5′-terminus by atleast 5 and at most 10 guanosine entities, and wherein said palindromicsequence is flanked at its 3′-terminus by at least 5 and at most 10guanosine entities. In a further preferred embodiment, the palindromicsequence, preferably SEQ ID NO:28, is flanked at its 3′-terminus by atleast 10, preferably exactly 10, guanosine entities and at its5′-terminus by at least 10, preferably exactly 10, guanosine entities.In a very preferred embodiment said ISS-NA is a A-type CpG comprising apalindromic sequence, wherein said palindromic sequence is flanked atits 5′-terminus by 3 to 10 guanosine entities, and wherein saidpalindromic sequence is flanked at its 3′-terminus by 3 to 10 guanosineentities. In a even more preferred embodiment said ISS-NA is a A-typeCpG comprising a palindromic sequence, wherein said palindromic is SEQID NO:28, and wherein said palindromic sequence is flanked at its5′-terminus by 3 to 10 guanosine entities, and wherein said palindromicsequence is flanked at its 3′-terminus by 3 to 10 guanosine entities.These immunostimulatory substances can be efficiently packaged intoVLPs, wherein the packaging efficiency is typically decreasing withincreasing number of flanking guanosine entities at the 5′ and/or 3′terminus.

In a very preferred embodiment of the present invention, theunmethylated CpG-containing oligonucleotide comprises, or alternativelyconsists essentially of, or alternatively consists of the a sequenceselected from the group consisting of (a) “G8-8” GGGGGGGG GACGATCGTCGGGGGGGG (SEQ ID NO:25); (b) “G9-9” GGGGGGGGG GACGATCGTC GGGGGGGGG (SEQID NO:26); or (c) “G10” GGGGGGGGGG GACGATCGTC GGGGGGGGGG (SEQ ID NO:27).The latter was previously found to be able to stimulate blood cells invitro (Kuramoto E. et al., Japanese Journal Cancer Research 83,1128-1131 (1992)).

In a specifically preferred embodiment the unmethylated CpG-containingoligonucleotide comprises, or alternatively consists essentially of, oralternatively consists of “010” (SEQ ID NO:27), wherein preferably saidG10 consists exclusively of phosphodiester bound nucleotides, whereinfurther preferably said G10 is aggregated G10 having a retention timerelative to Qβ capsid standard under HPLC conditions as set forth inExample 2 of 80 to 120%, most preferably of 80 to 95%.

In a further specifically preferred embodiment the unmethylatedCpG-containing oligonucleotide comprises, or alternatively consistsessentially of, or alternatively consists of “G9-9” (SEQ ID NO:26). In afurther specifically preferred embodiment the unmethylatedCpG-containing oligonucleotide comprises, or alternatively consistsessentially of, or alternatively consists of “G8-8” (SEQ ID NO:25).

In a further preferred embodiment said ISS-NA is an unmethylatedCpG-containing oligonucleotide, wherein the CpG motif of saidunmethylated CpG-containing oligonucleotide is part of a palindromicsequence, wherein said unmethylated CpG-containing oligonucleotide has anucleic acid sequence selected from (a) “G3-6” GGG GACGATCGTC GGGGGG(SEQ ID NO:29); (b) “G4-6” GGGG GACGATCGTC GGGGGG (SEQ ID NO:30); (c)“G5-6” GGGGG GACGATCGTC GGGGGG (SEQ ID NO:31); (d) “G6-6” GGGGGGACGATCGTC GGGGGG (SEQ ID NO:32); and (e) “G7-7” GGGGGGG GACGATCGTCGGGGGGG (SEQ ID NO:33); (f) “G8-8” GGGGGGGG GACGATCGTC GGGGGGGG (SEQ IDNO:25); (g) “G9-9” GGGGGGGGG GACGATCGTC GGG GGGGG (SEQ ID NO:26); and(h) “G6” GGGGGG CGACGACGATCGTCGTCG GGGGGG (SEQ ID NO:34).

In a further preferred embodiment of the present invention the ISS-NA isan unmethylated CpG-containing oligonucleotide, wherein the CpG motif ofsaid unmethylated CpG-containing oligonucleotide is part of apalindromic sequence, wherein said palindromic sequence is GACGATCGTC(SEQ ID NO:28), and wherein said palindromic sequence is flanked at its5′-terminus of at least 4 and at most 9 guanosine entities and whereinsaid palindromic sequence is flanked at its 3′-terminus of at least 6and at most 9 guanosine entities.

In another preferred embodiment of the present invention theimmunostimulatory substance is an unmethylated CpG-containingoligonucleotide, wherein the CpG motif of said unmethylatedCpG-containing oligonucleotide is part of a palindromic sequence,wherein said unmethylated CpG-containing oligonucleotide has a nucleicacid sequence selected from (a) “G4-6” GGGG GACGATCGTC GGGGGG (SEQ IDNO:30); (b) “G5-6” GGGGG GACGATCGTC GGGGGG (SEQ ID NO:31); (c) “G6-6”GGGGGG GACGATCGTC GGGGGG (SEQ ID NO:32); (d) “G7-7” GGGGGGG GACGATCGTCGGGGGGG (SEQ ID NO:33); (e) “08-8” GGGGGGGG GACGATCGTC GGGGGGGG (SEQ IDNO:25); and (f) “G9-9” GGGGGGGGGG GACGATCGTC GGGGGGGGG (SEQ ID NO:26).

In another preferred embodiment of the present invention the ISS-NA isan unmethylated CpG-containing oligonucleotide, wherein the CpG motif ofsaid unmethylated CpG-containing oligonucleotide is part of apalindromic sequence, wherein said unmethylated CpG-containingoligonucleotide has a nucleic acid sequence selected from (a) “G4-6”GGGG GACGATCGTC GGGGGG (SEQ ID NO:30); (b) “G5-6” GGGGG GACGATCGTCGGGGGG (SEQ ID NO:31); (c) “G6-6” GGGGG GACGATCGTC GGGGGG (SEQ IDNO:32); (d) “G7-7” GGGGGGG GACGATCGTC GGGGGGG (SEQ ID NO:33); (e) “G8-8”GGGGGGGG GACGATCGTC GGGGGGGG (SEQ ID NO:25); and (f) “G9-9” GGGGGGGGGACGATCGTC GGGGGGGGG (SEQ ID NO:26).

In a further preferred embodiment of the present invention the ISS-NA isan unmethylated CpG-containing oligonucleotide, wherein the CpG motif ofsaid unmethylated CpG-containing oligonucleotide is part of apalindromic sequence, wherein said palindromic sequence is GACGATCGTC(SEQ ID NO:28), and wherein said palindromic sequence is flanked at its5′-terminus of at least 5 and at most 8 guanosine entities and whereinsaid palindromic sequence is flanked at its 3′-terminus of at least 6and at most 8 guanosine entities.

The experimental data show that the ease of packaging said ISS-NA,preferably the guanosine flanked, palindromic and unmethylatedCpG-containing oligonucleotides, wherein the palindromic sequence isGACGATCGTC (SEQ ID NO:28), and wherein the palindromic sequence isflanked at its 3′-terminus and at its 5′-terminus by less than 10guanosine entities, into particles, preferably VLPs, increases if thepalindromic sequences are flanked by fewer guanosine entities. However,decreasing the number of guanosine entities flanking the palindromicsequences leads to a decrease of stimulating blood cells in vitro. Thus,packagability is paid by decreased biological activity of the indicatedinventive immunostimulatory substances. The preferred embodimentsrepresent, thus, a compromise between packagability and biologicalactivity.

In another preferred embodiment of the present invention the ISS-NA isan unmethylated CpG-containing oligonucleotide, wherein the CpG motif ofsaid unmethylated CpG-containing oligonucleotide is part of apalindromic sequence, wherein said unmethylated CpG-containingoligonucleotide has a nucleic acid sequence selected from (a) “G05-6”GGGGG GACGATCGTC GGGGGG (SEQ ID NO:31); (b) “06-6” GGGGG GACGATCGTCGGGGGG (SEQ ID NO:32); (c) “G7-7” GGGGGGG GACGATCGTC GGGGGG (SEQ IDNO:33); and (d) “G8-8” GGGGGGG GACGATCGTC GGGGGGGG (SEQ ID NO:25).

In a very preferred embodiment of the present invention the ISS-NA is anunmethylated CpG-containing oligonucleotide, wherein the CpG motif ofsaid unmethylated CpG-containing oligonucleotide is part of apalindromic sequence, wherein said unmethylated CpG containingoligonucleotide has the nucleic acid sequence of SEQ ID NO:25 (G8-8).

As mentioned above, the optimal sequence used to package into VLPs is acompromise between packagability and biological activity. Taking thisinto consideration, the G8-8 immunostimulatory substance is a furthervery preferred embodiment of the present invention since it isbiologically highly active while it is still reasonably well packaged.

In a further preferred embodiment said ISS-NA is a unmethylated CpCcontaining oligonucleotide, wherein said unmethylated CpC containingoligonucleotide is capable of inducing the production of IFN-alpha in acell, preferably in PBMCs, spleenocytes or human pDCs, and whereinfurther preferably said unmethylated CpC containing oligonucleotide isselected from: (a) T*C*G*T*C*G*T*T*T*T*G*T*T*T*T*T*C*G*T*T*TG*T*C*G*T(2006-PS, SEQ ID NO: 76); (b) GGGGGACGAT CGTCGGGGGGG (2216-PO, SEQ IDNO:77); (c) G*G*GGGACGATCGTC*G*G*G*G*G*G (2216-PO core, SEQ ID NO:77);(d) GGTGCATCGATGCAGGGGGG (D19-PO, SEQ ID NO:78); (e)G*G*TGCATCGATGCAG*G*G*G*G*G (D19-PO core, SEQ ID NO:78); (f)GGGGACGATCGTCGGGGGG (G3-6, SEQ ID NO:29); (g) GGGGGACGATCGTCGGGGGG(G4-6, SEQ ID NO:30); (h) GGGGGGACGATCGTCGGGGGG (G5-6, SEQ ID NO:31);(i) GGGGGGGACGATCGTCGGGGGG (G6-6, SEQ ID NO:32); (j) GGGGGGGGACGATCGTCGGGGGGG (G7-7, SEQ ID NO:33); (k) GGGGGGGGGA CGATCGTCGG GGGGGG(G8-8, SEQ ID NO:25); (l) GGGGGGGGGGACGATCGTCGGGGGGGGG (G9-9, SEQ IDNO:26); (m) GGGGGGGGGGGACGATCGTCGGGGGGGGG (G10, SEQ ID NO:27); and (n)GGGGGGGGGG GACGATCGTC GGGGGGGGGG GGGGGGGGGG GACGATCGTC GGGGGGGGGG(G102x, SEQ ID NO:79), wherein * indicates a phosphorothioatemodification, while all other nucleotides are phosphodiester bound. In amore preferred embodiment said unmethylated CpG containingoligonucleotide is capable of inducing the production of IFN-alpha inhuman pDCs, wherein preferably said unmethylated CpG containingoligonucleotide is T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T(2006-PS, SEQ ID NO: 76), wherein * indicates a phosphorothioatemodification, while all other nucleotides are phosphodiester bound. In astill more preferred embodiment said unmethylated CpG containingoligonucleotide is capable of inducing the production of IFN-alpha inPBMCs or spleen cells, wherein preferably said unmethylated CpGcontaining oligonucleotide is selected from the group consisting of: (a)GGGGGACGAT CGTCGGGGGG (2216-PO, SEQ ID NO:77); (b)G*G*GGGACGATCGTC*G*G*G*G*G (2216-PO core, SEQ ID NO:77); (c)GGTGCATCGATGCAGGGGGG (D19-PO, SEQ ID NO:78); (d)G*G*TGCATCGATGCAG*G*G*G*G*G (D19-PO core, SEQ ID NO:78); (e)GGGGACGATCGTCGGGGGG (G3-6, SEQ ID NO:29); (f) GGGGGACGATCGTCGGGGGG(G4-6, SEQ ID NO:30); (g) GGGGGGACGATCGTCGGGGGG (G5-6, SEQ ID NO:31);(h) GGGGGGGACGATCOTCGGGGGG (G6-6, SEQ ID NO:32); (i) GGGGGGGGACGATCGTCGGGGGGG (G7-7, SEQ ID NO:33); (j) GGGGGGGGGA CGATCGTCGG GGGGGG(G8-8, SEQ ID NO:25); (k) GGGGGGGGGGACGATCGTCGGGGGGGGG (G9-9, SEQ IDNO:26); (l) GGGGGGGGGGGACGATCGTCGGGGGGGGGG (G10, SEQ ID NO:27); and (m)GGGGGGGGGG GACGATCGTC GGGGGGGGGG GGGGGGGGGG GACGATCGTC GGGGGGGGGG(G102x, SEQ ID NO:79), wherein * indicates a phosphorothioatemodification, while all other nucleotides are phosphodiester bound. In avery preferred embodiment said unmethylated CpG containingoligonucleotide is GGGGGGGGGG GACGATCGTC GGGGGGGGGG GGGGGGGGGGGACGATCGTC GGGGGGGGGG (G102x, SEQ ID NO:79), wherein preferably allnucleotides are phosphodiester bound.

In a further preferred embodiment said ISS-NA is a C-type CpG, whereinpreferably said C-type CpG comprises a palindromic sequence, whereinfurther preferably said palindromic sequence is selected from any one ofthe sequences depicted in SEQ ID NOs:53 to 60. In a further preferredembodiment said C-type CpG is SEQ ID NO:64 or SEQ ID NO:65, whereinpreferably all nucleic acids of said C-type CpG are phosphorothioatebound. Further preferred C-type CpGs are selected from the groupconsisting of (a) TCpGTCGTTTT ACGGCGCCGTG CCG (SEQ ID NO:64); (b)TCGTCGTTTT ACpGGCpGCCpGTGCCG (SEQ ID NO:64); (c) TCGTCGTTTTACpGGCGCCpGTGCCG (SEQ ID NO:64); and (d) TCGTCpGTTTT ACpGGCGCCpGTGCCG(SEQ ID NO:64); wherein p indicates phosphodiester bounds while allother nucleotides are phosphorothioate bound. C-type CpGs selected fromthe group consisting of (a) TCGTCGTTTTCGGCGCGCGCCG (SEQ ID NO:66); (b)TCGTCGTTTTCGACGGCCGTCG (SEQ ID NO:67); (c) TCGTCGTTTTCCGGCGCGCCGG (SEQID NO:68); (d) TCGTCGTTTTCGGCGCGCGTCG (SEQ ID NO:69); (e)TCGGCGCGCGCCGTCGTCGTTT (SEQ ID NO:70); (f) TTGGCGCGCGCCGTCGTCGTTT (SEQID NO:71); (g) TCGTCGTTTTCGTCGGCCGCCG (SEQ ID NO:72); (h)TCGTCGTTTTCGGCTTTTGCCG (SEQ ID NO:73); (i) TCOTCTTTTTCGGCGCGTTTTTTT (SEQID NO:74); and (j) TCGTCGTTTTCGGCGGCCGCCG (SEQ ID NO:75) are potentinducers of IFN-alpha production (Vollmer et al. 2004, Eur. J. Immunol.43:351-262, p. 253, see Table I therein) and are thus specificallypreferred ISS-NA in the context of the invention.

One embodiment of the invention is a composition for use in a method oftreating or preventing hypersensitivity in an animal, preferably amammal, most preferably a human, the composition comprising a particleand an immunostimulatory nucleic acid, wherein said particle is packagedwith said immunostimulatory nucleic acid, and wherein preferably saidhypersensitivity is an allergy or a non-allergic hypersensitivity. Thecompositions and pharmaceutical compositions of the invention can beused in a therapeutic as well as in a prophylactic treatment.

In a preferred embodiment said hypersensitivity is selected from thegroup consisting of: (a) asthma, (b) rhinitis, (c) conjunctivitis, (d)rhinoconjuctivitis. (e) dermatitis, (f) urticaria, and (g) anaphylaxis.

In a further preferred embodiment said hypersensitivity is an allergy,wherein said allergy is preferably selected from IgE-mediated allergyand non IgE-mediated allergy. In a preferred embodiment saidhypersensitivity is asthma, preferably IgE-mediated asthma, wherein saidasthma can be intermittent or persistent asthma. In a very preferredembodiment said hypersensitivity is atopic asthma.

In a further preferred embodiment said hypersensitivity is dermatitis,preferably eczema, most preferably atopic eczema.

In a very preferred embodiment said hypersensitivity is an IgE mediatedallergy (type I allergy), wherein preferably said IRE-mediated allergyis an IgE-mediated allergy against a naturally occurring allergen, i.e.an allergen occurring in a natural source such as pollen, animal hair,house dust, dust mite etc.

In a preferred embodiment, said allergy, preferably said IgE-mediatedallergy, is selected from the group consisting of: (a) pollen allergy(hay fever), (b) house dust allergy, (c) food allergy, (d) drug allergy,(e) insect venom allergy, preferably bee venom allergy, and (f) animalallergy, preferably cat allergy.

In a further preferred embodiment said allergy, preferably saidIgE-mediated allergy, is an allergy against an allergen occurring in asource selected from the group consisting of (a) pollen: (b) dust,preferably house dust; (c) dust mite; (d) fungi, preferably aspergillus;(e) mammalian epideris, (f) bird feather; (g) insects, preferably beevenom; (h) food, preferably strawberry, kiwi, peanut, or wheat protein;(i) mammalian dander, preferably cat dander; (j) saliva; (k) serum; and(l) urine. In a further preferred embodiment said allergy, preferablysaid IgE-mediated allergy, is an allergy against an allergen occurringin a source selected from the group consisting of: (a) trees, (b)grasses, (c) animal products, and (d) plant products. In a furtherpreferred embodiment said allergy, preferably said IgE-mediated allergy,is an allergy against an antigen selected from the group consisting of(a) bee venom phospholipase A2; (b) ragweed pollen Amb a 1; (c) birchpollen Bet v I; (d) white faced hornet venom 5 Dol m V; (e) house dustmite Der p 1; (f) house dust mite Der f 2: (g) house dust mite DerP 2;(h) dust mite Lep d; (i) fungus allergen Alt a 1: (j) fungus allergenAsp f 1; (k) fungus allergen Asp f 16: and (l) peanut allergens.

In a further preferred embodiment said allergy, preferably saidIgE-mediated allergy, is an allergy against cat allergen, preferably anallergy against FelD1 antigen.

In a further preferred embodiment said allergy, preferably saidIgE-mediated allergy, is an allergy against dust mite, whereinpreferably said dust mite is selected from: (a) Dermatophagoidespteronyssinus, (b) D. farinae, (c) D. microceras, (d) Euroglyphusmaynei, (e) Glycyphagus sp., (f) Gohieria fusca, (g) Blomia tropicalis.

In a further preferred embodiment said allergy, preferably saidIgE-mediated allergy, is pollen allergy (hay fever). In furtherpreferred embodiment said allergy, preferably said IgE-mediated allergy,is house dust allergy, preferably IgE-mediated allergy against housedust mite allergens contained in house dust, wherein said house dustmite allergens are preferably selected from the group consisting of (a)Der p 1; (b) Der f 2; and (c) DerP 2.

The present invention, inter alia, relates to the finding thatparticles, preferably VLPs, can be packaged with ISS-NA, preferably withsingle stranded DNA oligonucleotides rich in non-methylated C and G(CpGs).

A preferred embodiment of the invention is therefore a composition foruse in a method of treating or preventing hypersensitivity in an animal,preferably a mammal, most preferably a human, the composition comprisinga VLP and an unmethylated CpG containing oligonucleotide, wherein saidVLP is packaged with said unmethylated CpG containing oligonucleotide.

A further preferred embodiment of the invention is a composition for usein a method of treating or preventing allergy in a human, thecomposition comprising a VLP of an RNA bacteriophage and an unmethylatedCpG containing oligonucleotide, wherein said VLP of an RNA bacteriophageis packaged with said unmethylated CpG containing oligonucleotide, andwherein preferably said unmethylated CpG-containing oligonucleotide isG10 (SEQ ID NO:27).

A very preferred embodiment of the invention is a composition for use ina method of treating or preventing allergy in a human, the compositioncomprising a VLP of RNA bacteriophage Qβ and unmethylated CpG containingoligonucleotide G10 (SEQ ID NO:27), wherein said VLP of RNAbacteriophage Qβ is packaged with said unmethylated CpG containingoligonucleotide G10.

Further embodiments of the invention are processes for the production ofthe compositions of the invention and methods for treatinghypersensitivity using said compositions, wherein preferably saidhypersensitivity is atopic asthma, type I allergy or atopic eczema.

The invention provides a process of producing a composition for use in amethod of treating hypersensitivity in an animal, said compositioncomprising a VLP and an unmethylated CpG containing oligonucleotide,wherein said VLP is packaged with said unmethylated CpG-containingoligonucleotide. said process comprising the steps of (i) incubatingsaid VIP (a) with said unmethylated CpG-containing oligonucleotide (b);(ii) adding RNase; and (iii) purifying said composition. In a preferredembodiment, said VLP is produced in a bacterial expression system. Inanother preferred embodiment, said RNase is RNase A.

In a further preferred embodiment, said process comprises the steps of(i) incubating said VLP with RNase; (ii) adding said unmethylatedCpG-containing oligonucleotide; and (iii) purifying the composition. Ina preferred embodiment, said VLP is produced in a bacterial expressionsystem. In another preferred embodiment, said RNase is RNase A.

In a further preferred embodiment, said process comprising the steps of(i) disassembling said VLP; (ii) adding said unmethylated CpG-containingoligonucleotide; and (iii) reassembling said VLP. In a furtherembodiment said process further comprises the step of removing nucleicacids from the disassembled VLP. Alternatively, said process furthercomprises the step of removing nucleic acids of the at least partiallydisassembled VLP and/or purifying the composition after reassembly. In apreferred embodiment, said VLP is produced in a bacterial expressionsystem. In a further preferred embodiment said VIP referred to in step(i) of said process is a VLP of an RNA bacteriophage, more preferably αVLP of an RNA bacteriophage selected from the group consisting of (a)bacteriophage Qβ, (b) bacteriophage AP205, (c) bacteriophage GA, and (d)bacteriophage fr. In a very preferred embodiment said VLP referred to instep (i) of said process is a VLP of a RNA bacteriophage, morepreferably a VLP of RNA bacteriophages AP205 or Qβ, most preferably ofQβ.

In a further preferred embodiment said unmethylated CpG-containingoligonucleotide referred to in step (ii) of said process consists of 5to 60 nucleotides, preferably of 20 to 40 nucleotides most preferably ofabout 30 nucleotides. In a very preferred embodiment said unmethylatedCpG-containing oligonucleotide is an A-type CpG, preferably an A-typeCpG comprising poly G motifs at the 5′ and/or 3′ ends. In a still morepreferred embodiment said unmethylated CpG-containing oligonucleotide isselected from the group consisting of: (a) “G8-8” GGGGGGGG GACGATCGTCGGGGGGGG (SEQ ID NO:25); (b) “G9-9” GGGGGGGGG GACGATCGTC GGGGGGGGG (SEQID NO:26); or (c) “G10” GGGGGGGGG GACGATCGTC GGGGGGGGG (SEQ ID NO:27),most preferably SEQ ID NO:27.

In a further preferred embodiment said process comprises the steps of(i) incubating said VLP in a solution comprising metal ions capable ofhydrolyzing the nucleic acids of said VLP; (ii) adding said unmethylatedCpG-containing oligonucleotide; and (iii) purifying said composition,wherein preferably said metal ions of step (i) are selected from thegroup consisting of (a) zinc (Zn) ions; (b) copper (Cu) ions; (c) iron(Fe) ions; (d) any mixtures of at least one ion of (a), (b) and/or (c).In a preferred embodiment, said VLP is produced in a bacterialexpression system.

In a further preferred embodiment said process comprises the steps of(i) incubating said VLP with solutions comprising metal ions capable ofhydrolyzing the nucleic acids of said VLP; (ii) adding said unmethylatedCpG-containing oligonucleotide; and (iii) purifying said composition,wherein preferably said metal ions of step (i) are selected from thegroup consisting of (a) zinc (Zn) ions; (b) copper (Cu) ions; (c) iron(Fe) ions; (d) magnesium (Mg) ions, and any mixtures of at least one ionof (a), (b), (c) and/or (d). In a preferred embodiment, said VLP isproduced in a bacterial expression system.

In a further preferred embodiment said process comprises the steps of(i) incubating said VLP with a solution capable of destabilizing saidVLP; (ii) purifying the coat protein from said solution; and (iii)reassembling said VLP in the presence of unmethylated CpG-containingoligonucleotide, wherein preferably said solution capable ofdestabilizing said VLP comprises magnesium chloride, wherein furtherpreferably the concentration of said magnesium chloride is 0.2 to 1.5M,more preferably 0.4 to 1M, most preferably about 0.7M; and wherein stillfurther preferably said VLP is a VLP of bacteriophage Qβ. In a verypreferred embodiment said process comprises the steps of (i) incubatingsaid VLP with a solution capable of destabilizing said VLP; (ii)purifying the coat protein from said solution; and (iii) reassemblingsaid VLP in the presence of unmethylated CpG-containing oligonucleotide,wherein preferably said solution capable of destabilizing said VLPcomprises magnesium chloride, wherein further preferably theconcentration of said magnesium chloride is 0.2 to 1.5M, more preferably0.4 to 1M, most preferably about 0.7M; and wherein still furtherpreferably said VLP is a VLP of bacteriophage Qβ; and wherein stillfurther preferably said unmethylated CpG-containing oligonucleotide isG10 (SEQ ID NO:27), most preferably said unmethylated CpG-containingoligonucleotide is aggregated G10 having a retention time relative to Qβcapsid standard under HPLC conditions as set forth in Example 2 of 80 to120%, most preferably of 80 to 95%.

In a further preferred embodiment said process comprises the steps of(i) incubating said VLP under alkaline conditions, preferably in thepresence of NaOH, most preferably in the presence of about 25 mM NaOH;(ii) adding said unmethylated CpG-containing oligonucleotide: and (iii)purifying said composition.

It was found that the efficiency of said reassembling of said VLP can beimproved and also that the stability of the reassembled VLP can beimproved, by subjecting the unmethylated CpG-containing oligonucleotideto conditions supporting the formation of oligonucleotide aggregatesprior to adding the oligonucleotide to the disassembled VLP. Conditionscontrolling the aggregation state of oligonucleotides have beendescribed in Guschlbauer W., Journal of Biomolecular Structure &Dynamics, ISSN 0739-1102, Volume 8, Issue Number 3 (1990). Title:Four-Stranded Nucleic Acid Structures 25 Years. The aggregation ofoligonucleotides is, for example, influenced by the ionic conditions,the concentration of the oligonucleotide, the pH, the temperatureconditions and by the incubation time. Furthermore, it was found thataggregation of the oligonucleotide is particularly advantages for A-typeoligonucleotides comprising poly G motifs at their 5′ and/or 3′ ends,most preferably for G10 (SEQ ID NO:27). Preferred conditions for theaggregation of unmethylated CpG-containing oligonucleotides areexemplified in Example 2. The optimal aggregation conditions may varybetween different unmethylated CpG-containing oligonucleotides and evenbetween different batches of the same unmethylated CpG-containingoligonucleotide. The actual aggregation state of an unmethylatedCpG-containing oligonucleotide can be assessed by HPLC, preferably underconditions as set forth in Example 2.

Thus, in a further embodiment the invention provides a processcomprising the steps of (i) disassembling said VLP; (ii) adding saidunmethylated CpG-containing oligonucleotide; and (iii) reassembling saidVLP, wherein prior to said adding of said unmethylated CpG-containingoligonucleotide said process comprises the additional step of incubatingsaid unmethylated CpG-containing oligonucleotide under conditionssupporting the formation of oligonucleotide aggregates. In a preferredembodiment said incubating is performed at a temperature of 70 to 100°C., preferably at about 85° C., preferably in the presence of sodiumions. In a still more preferred embodiment said incubating is performedat a concentration of said unmethylated CpG-containing oligonucleotideof 100 to 250 μm, preferably about 175 μm, in the presence of 200 to 500mM sodium ions, preferably in the presence of about 250 mM sodium ions,preferably at 85° C. for about 10 min, wherein further preferably saidunmethylated CpG-containing oliginucleotide comprises poly G motifs attheir 5′ and/or 3′ ends. In a still more preferred embodiment saidunmethylated CpG-containing oliginucleotide is selected from the groupconsisting of: (a) “08-8” GGGGGGGG GACGATCGTC GGGGGGGG (SEQ ID NO:25);(b) “G9-9” GGGGGGGGG GACGATCGTC GGGGGGGGG (SEQ ID NO:26); or (c) “G10”(GGGGGGGGGG GACGATCGTC GGGGGGGGGG (SEQ ID NO:27), most preferably saidunmethylated CpG-containing oliginucleotide is G10 (SEQ ID NO:27). In afurther preferred embodiment said conditions supporting the formation ofoligonucleotide aggregates are chosen in such a way that aggregatedunmethylated CpG containing oligonucleotide, preferably aggregated G10(SEQ ID NO:27) is obtained, wherein said aggregated unmethylated CpGcontaining oligonucleotide, preferably said aggregated G10 (SEQ IDNO:27) shows a retention time relative to Qβ capsid standard under HPLCconditions as set forth in Example 2 of 80 to 120%, most preferably of80 to 95%.

The invention further provides a process of producing a composition foruse in a method of treating hypersensitivity in an animal, saidcomposition comprising (a) a virus-like particle, a VLP of bacteriophageQβ; and (b) an unmethylated CpG-containing oligonucleotide; wherein saidvirus-like particle (a) is packaged with said unmethylatedCpG-containing oligonucleotide (b), said process comprising the steps of(i) incubating said VLP with a solution capable of destabilizing saidVLP; (ii) purifying the coat protein from said solution; and (iii)reassembling said coat protein to a VLP in the presence of unmethylatedCpG-containing oligonucleotide and an oxidizing agent. In a preferredembodiment, said solution capable of destabilizing said VLP comprisesmagnesium chloride and a reducing agent, wherein preferably theconcentration of said magnesium chloride is 0.2 to 1.5M, more preferably0.4 to 1M, most preferably about 0.7M; and wherein further preferablysaid reducing agent is DTT, and wherein still further preferably theconcentration of said DTT is 1 to 100 mM, preferably 2 to 15 mM, morepreferably about 10 mM and most preferably about 10 mM. In a furtherpreferred embodiment said oxidizing agent is H₂O₂, wherein furtherpreferably the concentration of said H₂O₂ is 1 to 50 mM, preferably 1 to10 mM, most preferably about 7 mM. In a further preferred embodimentsaid reassembling of said coat protein to a VLP is performed in thepresence of salt, wherein preferably said salt is NaCl, and whereinfurther preferably the concentration of said salt, preferably of saidNaCl is 100 mM to 1M, most preferably 100 mM to 500 mM, most preferablyabout 250 mM. In a further preferred embodiment, prior to saidreassembling said coat protein to a VLP, said purified coat protein isincubated in a solution comprising salt, reducing agent and unmethylatedCpG-containing oligonucleotide, wherein preferably (i) said salt isNaCl, and wherein further preferably the concentration of said salt,preferably of said NaCl is 100 mM to 1M, most preferably 100 mM to 500mM, most preferably about 250 mM; (ii) the concentration of said urea is100 mM to 7M, more preferably 500 mM to 2M, most more preferably about1M; and (iii) said reducing agent ist DTT, wherein preferably theconcentration of said DTT is 1 to 10 mM, preferably 1 to 5 mM, mostpreferably 2.5 mM. In a further preferred embodiment said unmethylatedCpG-containing oligonucleotide is G10 (SEQ ID NO:27), wherein mostpreferably said unmethylated CpG-containing oligonucleotide isaggregated G10 having a retention time relative to Qβ capsid standardunder HPLC conditions as set forth in Example 2 of 80 to 120%. mostpreferably of 80 to 95%.

The invention further provides compositions for use in a method oftreating hypersensitivity in an animal, wherein said compositions areobtainable by any one of the processes disclosed herein.

It is apparent for the artisan that the processes for the production ofa composition of the invention as described above can also be performedusing a virus particle instead of said VLP, wherein said virus particlepreferably is a virus particle of a bacteriophage, preferably of a RNAbacteriophage.

In particular, the invention provides compositions for use in a methodof treating hypersensitivity in an animal, wherein said compositions areobtainable by a process comprising the steps of (i) incubating a VLPwith unmethylated CpG-containing oligonucleotides; (ii) adding RNase;and (iii) purifying said composition. In a preferred embodiment, saidVLP is produced in a bacterial expression system. In another preferredembodiment, said RNase is RNase A.

The invention further provides compositions for use in a method oftreating hypersensitivity in an animal, wherein said compositions areobtainable by a process comprising the steps of (i) incubating a VLPwith RNase; (ii) adding unmethylated CpG-containing oligonucleotides;and (iii) purifying the composition. In a preferred embodiment, said VLPis produced in a bacterial expression system. In another preferredembodiment, said RNase is RNase A.

The invention further provides compositions for use in a method oftreating hypersensitivity in an animal, wherein said compositions areobtainable by a process comprising the steps of (i) disassembling a VLP;(ii) adding unmethylated CpG-containing oligonucleotides; and (iii)reassembling said VLP. In a further embodiment said process furthercomprises the step of removing nucleic acids from the disassembled VLP.Alternatively, said process further comprises the step of removingnucleic acids of the at least partially disassembled VLP and/orpurifying the composition after reassembly. In a preferred embodiment,said VLP is produced in a bacterial expression system.

The invention further provides compositions for use in a method oftreating hypersensitivity in an animal, wherein said compositions areobtainable by a process comprising the steps of (i) incubating a VLPwith solutions comprising metal ions capable of hydrolyzing the nucleicacids of said VLP; (ii) adding unmethylated CpG-containingoligonucleotides; and (iii) purifying said composition, whereinpreferably said metal ions of step (i) are selected from the groupconsisting of (a) zinc (Zn) ions; (b) copper (Cu) ions; (c) iron (Fe)ions: (d) any mixtures of at least one ion of (a), (b) and/or (c). In apreferred embodiment, said VLP is produced in a bacterial expressionsystem.

The invention further provides compositions for use in a method oftreating hypersensitivity in an animal, wherein said compositions areobtainable by a process comprising the steps of (i) incubating a VLPwith solutions comprising metal ions capable of hydrolyzing the nucleicacids of said VLP; (ii) adding unmethylated CpG-containingoligonucleotides; and (iii) purifying said composition, whereinpreferably said metal ions of step (i) are selected from the groupconsisting of (a) zinc (Zn) ions; (b) copper (Cu) ions; (c) iron (Fe)ions; (d) magnesium (Mg) ions; and (e) any mixtures of at least one ionof (a), (b), (c) and/or (d). In a preferred embodiment, said VLP isproduced in a bacterial expression system.

The invention further provides compositions for use in a method oftreating hypersensitivity in an animal, wherein said compositions areobtainable by a process comprising the steps of (i) incubating said VLPwith a solution capable of destabilizing said VLP, wherein preferablysaid VLP is a VLP of bacteriophage Qβ; (ii) purifying the coat proteinfrom said solution; and (iii) reassembling said coat protein to a VLP inthe presence of unmethylated CpG-containing oligonucleotide and anoxidizing agent. In a preferred embodiment, said solution capable ofdestabilizing said VLP comprises magnesium chloride and a reducingagent, wherein preferably the concentration of said magnesium chlorideis 0.2 to 1.5M, more preferably 0.4 to 1M, most preferably about 0.7M;and wherein further preferably said reducing agent is DTT, and whereinstill further preferably the concentration of said DTT is 1 to 100 mM,preferably 2 to 15 mM, more preferably about 10 mM and most preferablyabout 10 mM. In a further preferred embodiment said oxidizing agent isH₂O₂, wherein further preferably the concentration of said H₂O₂ is 1 to50 mM, preferably 1 to 10 mM, most preferably about 7 mM. In a furtherpreferred embodiment said reassembling of said coat protein to a VLP isperformed in the presence of salt, wherein preferably said salt is NaCl,and wherein further preferably the concentration of said salt,preferably of said NaCl is 100 mM to 1M, most preferably 100 mM to 500mM, most preferably about 250 mM. In a further preferred embodiment,prior to said reassembling said coat protein to a VLP, said purifiedcoat protein is incubated in a solution comprising salt, reducing agentand unmethylated CpG-containing oligonucleotide, wherein preferably (i)said salt is NaCl, and wherein further preferably the concentration ofsaid salt, preferably of said NaCl is 100 mM to 1M, most preferably 100mM to 500 mM, most preferably about 250 mM; (ii) the concentration ofsaid urea is 100 mM to 7 M, more preferably 500 mM to 2 M, most morepreferably about 1M; and (iii) said reducing agent ist DTT, whereinpreferably the concentration of said DTT is 1 to 10 mM, preferably 1 to5 mM, most preferably 2.5 mM. In a further preferred embodiment saidunmethylated CpG-containing oligonucleotide is G10 (SEQ ID NO:27),wherein most preferably said unmethylated CpG-containing oligonucleotideis aggregated G10 having a retention time relative to Qβ capsid standardunder HPLC conditions as set forth in Example 2 of 80 to 120%, mostpreferably of 80 to 95%.

The invention further provides compositions for use in a method oftreating hypersensitivity in an animal, wherein said compositions areobtainable by a process comprising the steps of (i) incubating said VLPunder alkaline conditions, preferably in the presence of NaOH, mostpreferably in the presence of about 25 mM NaOH; (ii) adding saidunmethylated CpG-containing oligonucleotide; and (iii) purifying saidcomposition.

The invention further provides compositions for use as a medicament,wherein said compositions are obtainable by any one of the processes ofthe invention, said composition comprising a particle and an ISS-NA,wherein said particle is packaged with said unmethylated CpG-containingoligonucleotide, wherein said particle preferably is a VLP of a RNAbacteriophage, most preferably of RNA bacteriophage Qβ, and whereinpreferably said ISS-NA is an unmethylated CpG-containingoligonucleotide, wherein said unmethylated CpG-containingoligonucleotide preferably exclusively consists of phosphodiester boundnucleotides, wherein further preferably said unmethylated CpG-containingoligonucleotide comprises the palindromic sequence of SEQ ID NO:28,wherein most preferably said unmethylated CpG-containing oligonucleotideis G10 (SEQ ID NO:27).

The invention further provides compositions for use in a method oftreating hypersensitivity, preferably allergy, most preferably atopicasthma, atopic eczema, pollen allergy, house dust or dust mite allergy,in an animal, wherein said compositions are obtainable by any one of theprocesses of the invention, said composition comprising (a) a VLP; and(b) an unmethylated CpG-containing oligonucleotide; wherein saidvirus-like particle (a) is packaged with said unmethylatedCpG-containing oligonucleotide (b), wherein said VLP preferably is a VLPof an RNA bacteriophage, most preferably of RNA bacteriophage Qβ. andwherein said unmethylated CpG-containing oligonucleotide preferablyexclusively consists of phosphodiester bound nucleotides, whereinfurther preferably said unmethylated CpG-containing oligonucleotidecomprises the palindromic sequence of SEQ ID NO:28, wherein mostpreferably said unmethylated CpG-containing oligonucleotide is G10 (SEQID NO:27).

The invention further provides compositions for use in a method oftreating hypersensitivity, preferably allergy, most preferably atopicasthma, atopic eczema, pollen allergy, house dust or dust mite allergy,in an animal, wherein said compositions are obtainable by any one of theprocesses of the invention, said composition comprising a VLP and anunmethylated CpG-containing oligonucleotide, wherein said virus-likeparticle is packaged with said unmethylated CpG-containingoligonucleotide, wherein said VLP preferably is a VLP of a RNAbacteriophage, most preferably of RNA bacteriophage Qβ, and wherein saidunmethylated CpG-containing oligonucleotide preferably exclusivelyconsists of phosphodiester bound nucleotides, wherein further preferablysaid unmethylated CpG-containing oligonucleotide comprises thepalindromic sequence of SEQ ID NO:28, wherein most preferably saidunmethylated CpG-containing oligonucleotide is G10 (SEQ ID NO:27).

The invention also provides pharmaceutical compositions for use in amethod of treating preventing and/or attenuating hypersensitivity in ananimal. Pharmaceutical compositions of the invention comprise, oralternatively consist of, an immunologically effective amount of theinventive compositions together with a pharmaceutically acceptablediluent, carrier or excipient. The pharmaceutical composition may alsooptionally comprise an adjuvant.

In a further embodiment said pharmaceutical composition comprises a slowrelease formulation of the composition of the invention. Slow releaseformulations are well known in the art. Typical and preferred slowrelease formulations are compositions of the invention formulated inmicroparticles, emulsions, and gels.

In one embodiment, the invention provides pharmaceutical compositionsfor treating or preventing atopic eczema. In another embodiment, theinvention provides pharmaceutical compositions for treating orpreventing asthma. In another embodiment, the invention providespharmaceutical compositions for treating or preventing IgE-mediatesallergy (type I allergy), preferably pollen allergy, house dust or dustmite allergy.

As would be understood by one of ordinary skill in the art, whencompositions of the invention are administered to an animal, they can bein a composition which contains salts, buffers, adjuvants or othersubstances which are desirable for improving the efficacy of thecomposition. Examples of materials suitable for use in preparingpharmaceutical compositions are provided in numerous sources includingREMINGTON'S PHARMACEUTCICAL SCIENCES (Osol, A, ed., Mack Publishing Co.,(1990)).

Various adjuvants can be used to increase the immunological response,depending on the host species, and include but are not limited to,Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Suchadjuvants are also well known in the art. Further adjuvants that can beadministered with the compositions of the invention include, but are notlimited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21,QS-18, CRL1005, Aluminum salts (Alum), MF-59, OM-174. OM-197. OM-294,isomatrix, and virosomal adjuvant technology. The adjuvants can alsocomprise a mixture of these substances.

Immunologically active saponin fractions having adjuvant activityderived from the bark of the South American tree Quillaja SaponariaMolina are known in the art. For example QS21, also known as QA21, is anHplc purified fraction from the Quillaja Saponaria Molina tree and it'smethod of its production is disclosed (as QA21) in U.S. Pat. No.5,057,540. Quillaja saponin has also been disclosed as an adjuvant byScott et al, Int. Archs. Allergy Appl. Immun., 1985, 77, 409.Monosphoryl lipid A and derivatives thereof are known in the art. Apreferred derivative is 3 de-o-acylated monophosphoryl lipid A, and isknown from British Patent No. 2220211. Further preferred adjuvants aredescribed in WO00/00462, the disclosure of which is herein incorporatedby reference.

Compositions of the invention are said to be “pharmacologicallyacceptable” if their administration can be tolerated by a recipientindividual. Further, the compositions of the invention will beadministered in a “therapeutically effective amount” (i.e., an amountthat produces a desired physiological effect).

The compositions of the present invention can be administered by variousmethods known in the art. The particular mode selected will depend ofcourse, upon the particular composition selected, the severity of thecondition being treated and the dosage required for therapeuticefficacy. The methods of the invention, generally speaking, can bepracticed using any mode of administration that is medically acceptable,meaning any mode that produces effective levels of the active compoundswithout causing clinically unacceptable adverse effects. Such modes ofadministration include oral, rectal, parenteral, intracistemal,intravaginal, intraperitoneal, topical (as by powders, ointments, dropsor transdermal patch), bucal, or as an oral or nasal spray. The term“parenteral” as used herein refers to modes of administration whichinclude intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion. The compositionof the invention can also be injected directly in a lymph node.Compositions comprising microparticles are preferably injectedsubcutaneously, intravenously, intradermaly, intraperitoneally,administered intranasally. orally, transdermally or inhaled.

Components of compositions for administration include sterile aqueous(e.g., physiological saline) or non-aqueous solutions and suspensions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Carriers or occlusive dressings can be used toincrease skin permeability and enhance antigen absorption.

Dosage levels depend on the mode of administration, the nature of thesubject, and the quality of the carrier/adjuvant formulation. Typicalamounts are in the range of about 0.001 μg to about 20 mg per subject.Preferred amounts are 50 μg to 1000 μg, more preferanly 100 μg to 600μg, and most preferably about 300 μg of a composition of the inventionper single administration. Further preferred amounts are at least about50 μg to about 500 μg per subject, most preferably 300 μg per subject.Multiple administration to treat the subject is preferred, and protocolsare those standard in the art adapted to the subject in question. Theadministration of said composition or said pharmaceutical composition isrepeated several times, preferably at least three to 10 times, mostpreferably three to five times, in weekly, monthly or yearly intervals,preferably in intervals of about 1 week to about 1 month, morepreferably in biweekly intervals, most preferably in weekly intervals.In a very preferred embodiment the administration of said composition orsaid pharmaceutical composition is repeated 6 times in weekly intervals,wherein preferably each time 50 μg to about 500 μg, most preferablyabout 300 μg are administered.

The compositions of the invention can conveniently be presented in unitdosage form and can be prepared by any of the methods well-known in theart of pharmacy. Methods include the step of bringing the compositionsof the invention into association with a carrier which constitutes oneor more accessory ingredients. In general, the compositions are preparedby uniformly and intimately bringing the compositions of the inventioninto association with a liquid carrier, a finely divided solid carrier,or both, and then, if necessary, shaping the product.

Compositions suitable for oral administration can be presented asdiscrete units, such as capsules, tablets or lozenges, each containing apredetermined amount of the compositions of the invention. Othercompositions include suspensions in aqueous liquids or non-aqueousliquids such as a syrup, an elixir or an emulsion.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compositions of the invention described above,increasing convenience to the subject and the physician. Many types ofrelease delivery systems are available and known to those of ordinaryskill in the art.

A further aspect of the invention is a method of treatinghypersensitivity, preferably atopic eczema, atopic asthma orIgE-mediated allergy (type I allergy), in an animal, preferably amammal, most preferably a human, said method comprising introducing intosaid animal (i) a composition comprising a particle and an ISS-NA,wherein said particle is packaged with said ISS-NA or (ii) apharmaceutical composition comprising an immunologically effectiveamount of the composition (i) together with a pharmaceuticallyacceptable diluent, wherein preferably said pharmaceutical composition(ii) further comprises an adjuvant. In a preferred embodiment saidcomposition (i) or said pharmaceutical composition (it) is introducedinto said animal subcutaneously, intramuscularly, intravenously,intranasally or directly into the lymph node. In a further preferredembodiment said introducing into said animal of the composition (i) orthe pharmaceutical composition (ii) is repeated at least twice,preferably at least three times, most preferably at least four times inintervals of 1 week to 3 months, preferably 1 week. In a still furtherpreferred embodiment said introducing into said animal of thecomposition (i) or the pharmaceutical composition (ii) is repeated 6times in intervals of about 1 week, wherein preferably each time about300 μg of said composition (i) are introduced.

A preferred embodiment of the invention is a method of treating allergyin an animal, said method comprising introducing into said animal acomposition comprising (a) a VLP; and (b) an unmethylated CpG-containingoligonucleotide; wherein said virus-like particle (a) is packaged withsaid unmethylated CpG-containing oligonucleotide (b), and wherein saidallergy preferably is atopic eczema, asthma or type I allergy,preferably pollen allergy (hay fever), wherein further said VLP is a VLPof a RNA bacteriophage, preferably of RNA bacteriophage Qβ, and whereinpreferably said unmethylated CpG-containing oligonucleotide (b) consistsexclusively of phosphodiester bound nucleotides, most preferably of SEQID NO:27.

The effectiveness of a treatment of the invention with respect to aparticular disease can be assessed by assessing the severity of thesymptoms associated with said disease using standard methods known inthe art. Generally symptoms are scored directly before the beginning ofthe treatment, i.e. before the first vaccination, in intervals duringthe treatment and 1 to 3 months after the last treatment. Symptoms ofatopic dermatitis can, for example be scored as described in N. Engl. J.Med 1997, 337:816-21. Symptoms of asthma can be scored by variousmethods including questionnaires described in Juniper et al., HealthQual. Life Outcomes, 2005 Sep. 16, 3:58, and combinations ofquestionnaires and spirometric measurements of pulmonary functions asdescribed in N. Engl. J. Med 2000, 343:1054-63. These references areincorporated herein by reference. Pollen allergy can, inter alia, beassessed using a nasal provocation test, other allergies, e.g. housedust or dust mite allergy, can be assessed using a conjunctivalprovocation procedure or a skin prick test. These testing methods aredescribed in detail in the Example section.

A further aspect of the invention is the use of a composition of theinvention or of a pharmaceutical composition of the invention for themanufacture of a pharmaceutical for the treatment of hypersensitivity inan animal, wherein said hypersensitivity preferably is an allergy,wherein further preferably said allergy is selected from the groupconsisting of: (a) atopic eczema; (b) atopic asthma; and (c)IgE-mediated allergy (type I allergy), preferably pollen allergy (hayfever) or house dust allergy.

A preferred embodiment of the invention is the use of a compositioncomprising a particle and an ISS NA, wherein said particle is packagedwith said ISS-NA, for the manufacture of a pharmaceutical for thetreatment of hypersensitivity in an animal, wherein hypersensitivity ispreferably selected from the group consisting of: (a) atopic eczema; (b)atopic asthma; and (c) IgE-mediated allergy (type I allergy), preferablypollen allergy (hay fever), and wherein further preferably said particleis a VLP of a RNA bacteriophage, preferably of RNA bacteriophage Qβ, andwherein preferably said ISS-NA is an unmethylated CpG-containingoligonucleotide, wherein preferably said unmethylated CpG-containingoligonucleotide consists exclusively of phosphodiester boundnucleotides, and wherein more preferably said unmethylatedCpG-containing oligonucleotide comprises the palindromic sequence of SEQID NO:28, most preferably said unmethylated CpG-containingoligonucleotide is G10 (SEQ ID NO:27).

In all aspects and embodiments, in particular in all compositions,methods, processes and uses, of the invention wherein said particle is aVLP of bacteriophage Qβ, said VLP preferrably essentially consists ofcoat proteins having the amino acid sequence of SEQ ID NO:3. In allaspects and embodiments, in particular in all compositions, methods,processes and uses, of the invention wherein said ISS-NA is theunmethylated CpG-containing oligonucleotide G10 (SEQ ID NO:27), saidunmethylated CpG-containing oligonucleotide preferrably consistsexclusively of phosphodiester bound nucleotides.

In all aspects and embodiments, in particular in all compositions,methods, processes and uses, of the invention wherein said particle is aVLP of bacteriophage Qβ and wherein said ISS-NA is the unmethylatedCpG-containing oligonucleotide 010 (SEQ ID NO:27), said VLP ofbacteriophage Qβ preferrably essentially consists of coat proteinshaving the amino acid sequence of SEQ ID NO:3, and, further preferably,said unmethylated CpG-containing oligonucleotide consists exclusively ofphosphodiester bound nucleotides.

The following examples are illustrative only and are not intended tolimit the scope of the invention as defined by the appended claims. Itwill be apparent to those skilled in the art that various modificationsand variations can be made in the methods of the present inventionwithout departing from the spirit and scope of the invention. Thus, itis intended that the present invention cover the modifications andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

If not indicated otherwise, the VLPs of bacteriophage Qβ used in theExamples are/were VLPs essentially consisting of coat proteins havingthe amino acid sequence of SEQ ID NO:3. Furthermore, if not indicatedotherwise, the unmethylated CpG containing oligonucleotide G10 (SEQ IDNO:27) used in the Examples is/was unmethylated CpG containingoligonucleotide G10 (SEQ ID NO:27) consisting exclusively ofphosphodiester bound nucleotides.

All patents, patent applications and publications referred to herein areexpressly incorporated by reference in their entirety.

EXAMPLES Example 1 Example for Packaging CpG-DNA 1668 into AP205 VLP andQβ VLP

Bacterial produced VLPs contain high levels of single stranded RNA,randomly packaged into the VLP during self assembly, which can bevisualized on a 1% agarose gel stained with ethidium bromide orCoomassie blue for the detection of RNA/DNA or protein. The RNA has tobe removed from the VLP before packaging with CpG DNA by digestion withRNase A. Therefore 1 mg/ml VLP in 20 mM HEPES, pH 7.4 was incubated with300 μg RNase A for 3 h at 37° C. For removal of RNAse A (and hydrolysedRNA) the VLP were dialysed for 2-3 days against 20 mM HEPES using a100,000 or 300,000 MW cut off membrane. Emptied VLPs were thensupplemented with CpG-oligonucleotide 1668-CpG (TCC ATG ACG TTC CTG AATAAT, SEQ ID NO:80) which was protected by a phosphorothioate backbone (1ml VLP (1 mg/ml in 20 mM HEPES, pH 7.4, 2 mM MgCl₂) with 100 nmol CpG)allowing free diffusion into the particle during incubation for 3 hrs at37° C. The CpG-oligonucleotide containing VLPs were purified fromunbound CpG-oligonucleotide via tangential flow filtration using a300,000 MW cut off membrane. A 1% agarose gel stained with ethidiumbromide or Coomassie blue visualized the removal of RNA followed bypackaging of CpG into VLP and proves the removal of unbound CpG.

Example 2 Disaggregation and Aggregation of Oligonucleotide G10 (SEQ IDNO:27) and Analysis of Aggregation State

Disaggregation (10.0 ml scale. 260 μM G10, 25 mM NaOH, 50° C. 70 min):45.91 mg G10 were weighed into a 15 ml tube. The powder was dissolved in11.0 ml purified water (c=325.3 μM; determined with the photometer). 8.0ml of the oligonucleotide solution were mixed with 250 μl 1M NaOH and1.75 ml purified water in a 15 ml tube (260 μM G10, 25 mM NaOH). Themixture was disaggregated for 70 minutes at 50° C. in a water bath.After cooling the solution on ice, the pH was adjusted with 0.5M HCl topH 5.31; 540 μl 0.5 M HCl and 5 μl 1M NaOH were added.

Aggregation (10.0 ml scale, 175 μM G10, 250 mM NaOH, 85° C., 9-24 min):7.1 ml disaggregated G10 solution, 2.13 ml purified water and 770 μl 3 MNaCl were mixed in a 15 ml tube (175 μM oligo, 250 mM Na⁺). The mixturewas incubated for 9 minutes at 85° C. in a water bath. The solution wascooled down in an ice/water bath and stored on ice until use. Aggregatedoligonucleotide solutions should be used within 3 hours afterpreparation.

Quantification of G10: G10 was quantified by UV absorption at 260 nmcorrected by the absorption at 340 nm. 1 A₂₆₀₋₃₄₀=27.8 μg/ml.

Analysis of aggregation state: The aggregation state of G10 was analysedby HPLC using the following conditions using Qβ capsid as standard.

Column: TSKgel 5000 PWXL 7.8 mm*30.0 cm

-   -   (Lot: 5PWX06GNMH3304, Art: 08023, Tosoh Bioscience)

Eluent: PBS pH 7.2

Injection volume: 40.0 μl

Flow rate: 0.8 ml/min

Gradient: Isocratic

Run time: 20 min

Wavelength: 215, 260 and 280 nm, data evaluation at 260 nm

Column oven temp.: 25° C.

Autosampler temp.: 8° C.

The relative retention time X % of G10 was calculated as follows: X%=peak start time [min]/Retention time (Qβ capsid) [min]×100%.Disaggregated G10 showed a relative retention time of 138%(136.9-140.3%; n=5). G10 preparations which have not undergone thedisaggregation/aggregation treatment described above show a relativeretention time in the same range. After disaggregation and aggregation,the relative retention time of G10 was found to be 118%.

Example 3 Packaging of Qβ VLPs with G10 by Disassembly/Reassembly

Disassembly of Qβ VLPs: 45 mg Qβ VLP (2.5 mg/ml, as determined byBradford analysis) in PBS (20 mM Phosphate, 150 mM NaCl, pH 7.5), wasreduced with 10 mM DTT for 15 min at RT under stirring conditions. Then,magnesium chloride was added to 0.7M final concentration and theincubation was continued for 15 min at RT under stirring conditions,leading to precipitation of the encapsulated host cell RNA andconcomitant disintegration of the VLPs. The solution was centrifuged 10min at 4000 rpm at 4° C. (Eppendorf 5810 R, in fixed angle rotor A-4-62used in all following steps) in order to remove the precipitated RNAfrom the solution. The supernatant, containing the released, dimeric Qβcoat protein, was used for the chromatographic purification steps.

Purification of Qβ coat protein by cation exchange chromatography andsize exclusion chromatography: The supernatant of the disassemblyreaction, containing dimeric coat protein, host cell proteins andresidual host cell RNA, was loaded onto a SP-Sepharose FF column(xk16/20, 6 ml, Amersham Bioscience). The column was equilibrated with20 mM sodium phosphate buffer pH 7 and the sample was diluted 1:15 inwater to adjust a conductivity below 10 mS/cm in order to achieve properbinding of the coat protein to the column. The elution of the bound coatprotein was accomplished by a step gradient to 20 mM sodiumphosphate/500 mM sodium chloride and the protein was collected in afraction volume of approx. 25 ml. The chromatography was carried out atRT with a flow rate of 5 ml/min during all steps and the absorbance wasmonitored at 260 nm and 280 nm. In a second step, the isolated Qβ coatprotein (the eluted fraction from the cation exchange column) was loadedonto a Sephacryl S-100 HR column (xk26/60, 320 ml, Amersham Bioscience)equilibrated with 20 mM sodium phosphate/250 mM sodium chloride; pH 7.2.The chromatography was carried out at RT with a flow rate of 2.5 ml/minand the absorbance was monitored at 260 nm and 280 nm. Fractions of 5 mlwere collected.

Characterization of purified Qβ coat protein by analytical sizeexclusion chromatography: A sample of purified Qβ coat protein wasanalyzed by analytical size exclusion chromatography (FIG. 1C) andcompared to i) intact Qβ VLP (FIG. 1A), which had been purified from E.coli lysate and which was used as source material for the purificationprocedure, and ii) to the supernatant of the disassembly reaction (FIG.1B). Efficient separation of RNA molecules from the coat protein isindicated by the absence of any RNA-like peak (typical ratio ofA280/A260=0.5) in FIG. 1C and the presence of a unique protein-like peak(typical ratio of A280/A260=1.7).

Assembly of Qβ G10 by diafiltration: Purified coat protein (in 20 mMsodium phosphate pH 7.2, 250 mM NaCl) was mixed with water and stocksolutions of urea, NaCl, DTT and aggregated G10 oligonucleotide(prepared essentially as described in Example 2). The volume of themixture was 50 ml and the final concentrations of the components were 1mg/ml coat protein, 1.0 M urea, 250 mM NaCl, 2.5 mM DTT and 0.24 mg/mlG10. The solution was then diafiltrated at room temperature against 300ml of 20 mM sodium phosphate 250 mM NaCl pH 7.2, using a 30 kDa cut offcartridge (Pellicon XL, Millipore) and a cross flow rate of 10 ml/minand a permeate flow rate of 2.5 ml/min. H₂O₂ was added to 7 mM finalconcentration and the solution incubated for 1 h at RT in order toinduce the formation of disulfide bonds. The solution was thendiafiltrated against 500 ml of 20 mM sodium phosphate 150 mM NaCl pH7.2, using a 300 kDa cut off cartridge (Pellicon XL, Millipore) and across flow rate of 10 ml/min and a permeate flow rate of 2.5 ml/min, inorder to remove excess of H₂O₂ and non-packaged G10 oligonucleotidesfrom the assembled QβG10 product.

Example 4 Analysis of QβG10 Packaging Product and Determination of Yieldof the Packaging Process

Characterization of packaged QβG10 VLP by analytical size exclusionchromatography: A sample of packaged QβG10 VLP was analyzed byanalytical size exclusion chromatography (FIG. 2) and compared to intactQβ VLP, which had been purified from E. coli lysate. The presence ofcorrectly assembled VLP in the product was confirmed by a peak migratingat identical retention time as the peak representing native Qβ VLP. Theobserved peak for QβG10 VLP (FIG. 2D) is dominated by the nucleic acidcontent of the VLP, because the absorption coefficient nucleic acids at260 nm is more than 100-fold higher than the absorption coefficient ofthe coat protein. The ratio A260/A280 of purified QβG10 VLP was found tobe 1.70 (1.65-1.76; n=5), which is characteristic for G10(A260/A280=1.74), wherein the A260/A280 ratio of Qβ VLP was found to be1.87 (1.85-1.90; n=10) which is characteristic for RNA.

Characterization of packaged QβG10 VLP by SDS-PAGE analysis: A sample ofpackaged Qβ G10 was analyzed by non-reducing SDS-PAGE (FIG. 3) andcompared to intact Qβ VLP, which had been purified from E. coli lysate.The presence of correctly assembled VLP in the product was confirmed bythe formation of bands of disulfide-linked pentameric and hexamericforms of the coat protein, similar to the intact Qβ VLPs, indicating thecorrect structural arrangement of the coat protein units in the in vitroassembled QβG10 VLP.

Quantification of packaged oligonucleotide G10: Samples of QβG10 VLP(0.25 mg/ml in PBS) were treated by 0.1 mM TCEP (15 min at RT) in orderto reduce the disulfide bonds. NaCl was added to the reduced samples (1Mfinal concentration) and the mixtures were incubated for 15 min at 60°C. in order to precipitate the protein component. After centrifugation,the resulting supernatants were incubated for 5 min at 95° C., cooled onice for 1 min and then the A260 value was measured. The concentration ofoligonucleotide G10 in the supernatants was calculated according to theformula:

c(G10) (mg/ml)=A₂₆₀×1.12×9600/344580, where:

1.12=correction factor for the salt content in the sample

9600=molecular mass of oligonucleotide G10

344580=specific molar absorption coefficient of oligonucleotide G10.

Typically, the amount of packaged oligonucleotide G10 was 0.2 mg per mgof Qβ coat protein.

G10 content of QβG10 VLP and yield calculation for the packagingreaction: Aggregated G10 was packaged into Qβ VLP by assembly/reassemblyof the VLP as described in Example 3. 953 mg G10 oligonucleotide wereintroduced for reassembly with 4000 mg purified Qβ dimer. The reactionyielded QβG10 comprising 20 μg G10 oligonucleotide per 100 μg protein(protein content determined by Bradford analysis or HPLC). The G10 yieldof the packaging reaction was 63% at a protein yield of 75%.

Example 5 Packaging of AP205 and GA355 VLPs with G10 byDisassembly/Reassembly

Disassembly: 50-100 mg of AP205 or GA355 VLPs (as determined by Bradfordanalysis) in buffer A (5 mM NaPO₄ pH 6.8, 100 mM NaCl, 2 mM MgCl₂) wereincubated at 30° C. for 16 hours with RNAse A (Sigma) and Benzonase(Novagen) at 1 mg/ml and 5 U/ml, respectively. In the case of AP205 VLPdeoxidation of the internal disulfide bridges was performed precedingthe addition of RNAse A and Benzonase by addition of 20 mM DTT followedby a 30 min incubation at 37° C. After addition of 1M NaCl precipitationof the viral coat proteins was induced by 15 min incubation at 70° C.Precipitated coat proteins were sedimented by centrifugation for 10 min,27,000 g at 4° C. The supernatant containing RNAse A, Benzonase anddegraded nucleic acids was discarded. Pellets were resuspended in bufferB (20 mM NaPO₄ pH 7.2, 6M urea) and incubated for 10 min at roomtemperature.

Purification of coat proteins by cation exchange chromatography: Thesolutions were clarified by centrifugation for 10 min, 27,000 g at 4° C.A negligible pellet was discarded. And the supernatant containing thedisassembled coat proteins were applied on a SP Sepharose™ FF column(16/20, Amersham Biosciences) equilibrated with buffer B. The flowthrough was discarded. After an extensive wash with buffer B (15 CV) thecolumn was adjusted with a linear gradient from buffer B to buffer C (20mM NaPO₄ pH 7.2, 1M urea) with a gradient length of 37.5 CV. During theloading, wash and elution the absorbance at 254 nm and 280 nm wasmonitored. Coat proteins were eluted as one fraction with buffer D (20mM NaPO₄ pH 6.5, 1M urea, 300 mM NaCl) and analyzed by LDS-PAGE followedby Coomassie staining. Eluted protein fractions were stored at 4° C. as“disassembled coat protein”. Protein concentrations were determined byBradford analysis.

Reassembly: Purified AP205 or GA355 coat protein with were used in afive fold excess (w/w) to G10 oligonucleotide. The coat proteins weremixed with the G10 oligonucleotide in a reassembly buffer containing 1Murea and 2.5 mM DTT and incubated for one hour at room temperature.After incubation the reassembly mix was dialyzed for 24 hours against 5liter PBS. The resulting suspension was centrifuged for 10 min, 27.000 gat 4° C. A negligible sediment was discarded. The supernatant containedthe reassembled and packaged VLPs. Protein concentration was determinedby Bradford analysis and the reassembled and packaged VLPs wereconcentrated with centrifugal filter devices (Amicon Ultra 15, 10KMWCO).

Purification of reassembled and packaged VLPs; Up to 25 mg total proteinwas loaded onto a Sepharose™ CL-4B (26/60, Amersham Biosciences)equilibrated with PBS. Size exclusion chromatography was performed withequilibration buffer at room temperature with a flow rate of 1.25ml/min. During the elution absorbance at 254 nm and 260 nm wasmonitored. Two peaks were isolated. A major high molecular weight peakpreceded a small peak of lower apparent molecular weight. The major peakrevealed a apparent molecular weight consistent to purified VLPs asshown by SE-HPLC. Analysis of AP205 or GA355 VLPs packaged with G10oligonucleotide is performed essentially as shown in Example 16 ofWO03/024481 (p. 131 ff).

Example 6 Packaging of FR VLPs with G10 by Disassembly/Reassembly

Disassembly: 50-100 mg of FR VLPs (as determined by Bradford analysis)in buffer A (5 mM NaPO₄ pH 6.8, 100 mM NaCl, 2 mM MgCl₂) are incubatedat 30° C. for 16 hours with RNAse A (Sigma) and Benzonase (Novagen) at 1mg/ml and 5 U/ml, respectively. After addition of 1M NaCl precipitationof the FR coat proteins is induced by a 15 min incubation at 70° C.Precipitated coat proteins are sedimented by centrifugation for 10 min,27,000 g at 4° C. The supernatant containing RNAse A. Benzonase anddegraded nucleic acids are discarded. The pellet is resuspended inbuffer B (20 mM NaPO₄ pH 7.2, 6 M urea) and incubated for 10 min at roomtemperature.

Purification of FR coat proteins by cation exchange chromatography: Thesolution is clarified by centrifugation for 10 min, 27,000 g at 4° C. Anegligible pellet is discarded and the supernatant containing thedisassembled coat proteins is applied on a SP Sepharose™ FF column(16/20, Amersham Biosciences) equilibrated with buffer B. The flowthrough is discarded. After an extensive wash with buffer B (15 CV) thecolumn is adjusted with a linear gradient from buffer B to buffer C (20mM NaPO₄ pH 7.2, 1M urea) with a gradient length of 37.5 CV. During theloading, wash and elution the absorbance at 254 nm and 280 nm ismonitored. FR coat proteins are eluted as one fraction with buffer D (20mM NaPO₄ pH 6.5, 1M urea, 300 mM NaCl) and analyzed by LDS-PAGE followedby Coomassie staining. The eluted protein fractions is stored at 4° C.as “disassembled coat protein”. Protein concentration is determined byBradford analysis.

Reassembly; Purified FR coat protein is used in a five fold excess (w/w)to G10 oligonucleotide. The FR coat proteins are mixed with the 010oligonucleotide in a reassembly buffer containing 1M urea and 2.5 mM DTTand incubated for one hour at room temperature. After incubation thereassembly mix is dialyzed for 24 hours against 5 liter PBS. Theresulting suspension is centrifuged for 10 min, 27,000 g at 4° C. Anegligible sediment is discarded. The supernatant contains thereassembled and packaged FR VLPs. Protein concentration is determined byBradford analysis and the reassembled and packaged FR VLPs areconcentrated with centrifugal filter devices (Amicon Ultra 15, 10KMWCO).

Purification of reassembled and packaged FR VLPs: Up to 25 mg totalprotein is loaded onto a Sepharose™ CL-4B (26/60, Amersham Biosciences)equilibrated with PBS. Size exclusion chromatography is performed withequilibration buffer at room temperature with a flow rate of 1.25ml/min. During the elution absorbance at 254 nm and 260 nm is monitored.Two peaks are isolated. A major high molecular weight peak precedes asmall peak of lower apparent molecular weight. The major peak reveals aapparent molecular weight consistent to purified FR VLPs as shown bySE-HPLC. Analysis of FR VLPs packaged with G10 oligonucleotide isperformed essentially as shown in Example 16 of WO 03/024481 (p. 131ff).

Example 7 Size Dependent Traffic of Nanoparticles to the Lymph Node

To study the mechanism of traffic of particulate antigens to the lymphnode, yellow-green fluorescent polystyrene nanoparticles with sizesranging from 20 nm to 2000 nm (Molecular probes) were injected in thefootpads (25 μg/footpads) of C57BL/6 mice and tracked. VLPs ofbacteriophage Qβ were coupled to Alexa488 using a protein labeling kit(Molecular probes) and included in the experiment as 30 nm fluorescentparticles. Forty eight hours later the nanoparticles and VLPs weretracked in the popliteal draining lymph node (LN) by flow cytometry.Popliteal LN were isolated and digested with 1 mg/ml Collagenase D(Boehringer) and 0.04 mg/ml DNA-se I (Roche) for 30 min at 37° C. Inorder to identify cell populations taking up nanoparticles, LN cellswere stained with anti-CD11c-PE (BD). Flow cytometry analysis showedthat 48 hours after injection, nanoparticles and VLPs have reached thepopliteal LN and were associated to different level with LN cells,including dendritic cells (DC, Table 1). LN cells acquired moreefficiently smaller particles (20-500), than larger ones (1000-2000 nm).VLP-Alexa488 (30 nm) showed efficiency of LN uptake between theefficiency of 20 and 100 nm polystyrene nanoparticles. These datasuggest that nanoparticles and VLPs traffic from the injection site tothe LN in a size-dependent manner, with significant trafficking takingplace for particles of sizes between 20 and 500 nm.

TABLE 1 Flow cytometry analysis of the trafficking of nanoparticles orVLP to the popliteal LN: 48 h after injection. Anti-CD11c antibodieswere used to identify DC. Size [nm] % FL1 + DC sd % FL + LN cells sd 200.82 0.08 2.46 0.15 30 1.38 0.18 4.14 0.65 100 2.31 0.36 12.09 1.22

Example 8 Kinetics of Trafficking of VLP to the LN

To study the kinetics of uptake of nanoparticles in LN cells,VLP-Alexa488 (prepared as described in Example 7) were injected inC57BL/6 mice (as described in Example 7) and tracked after 2 to 96 h. LNcells were stained with combinations of the following antibodies:anti-CD11c-PE, anti-CD11b-APC, anti-CD11c-biotin, anti-CD19-APC,anti-B220-PE, antiCD8-CyChr, anti-CD40-APC and streptavidin-CyChr (allfrom BD). Flow cytometry analysis showed that 2 h after injection in thefootpad, VLP-Alexa488 have associated with antigen presenting cells(APC) from the popliteal LN (Table 2). It is quite unlikely thatdendritic cells from the skin had taken up and had traffickedVLP-Alexa488 to the draining LN for the short period of 2 h. Theseresults rather suggest that VLP-Alexa488 had drained in a cell-freemanner through the lymphatic system into the draining lymph node.Macrophages and DC were the most prominent populations taking upVLP-Alexa488. Most importantly, 2 h after injection, 1.9% of LN-residentplasmacytoid DC also acquired VLP-Alexa488. pDC in humans are mostlyfound in secondary lymphoid tissues and are the only DC populationexpressing TLR9. Therefore, the ability of VLP-Alexa488 to drain free tothe LN makes them a very appropriate vehicle to target pDC with thepossibility to simultaneously deliver ISS (such as CpG), packaged inVLP.

The uptake of VLP-Alexa488 peaked at 24 h after injection (Table 2),remained quite stable by 48 h and declined after 96 h. It is likely,that at that later time point also skin derived DC contribute to thetraffic of VLP-Alexa488 to the draining LN. Indeed, the ratio betweenthe uptake of VLP-Alexa488 at 2 h (draining) and 24 h (DC-mediatedtransport) is lower for myeloid (including skin-derived,CD11c+CD40hiCD8−) compared to lymphoid (CD11c+CD40loCD8+) DC, whichacquire VLP while in the LN (FIG. 4).

TABLE 2 Flow cytometry analysis of the kinetics of trafficking of Qβ-VLPto the popliteal LN. Anti-CD11c, CD11b, B220 and CD19 antibodies wereused to identify DC, Macrophages, pDC and B-cells. The percentage ofgreen cells in the gate of the indicated LN cell populations is shown. %VLP-Alexa488+ cells time [h] DCs Macrophages pDC B-cells 2 2.82 13.041.9 0.9 24 26.83 38.32 3.66 2 48 18.28 24.9 1.5 0.96 96 4.31 18.35 0.240.21

Example 9 Kinetics of Trafficking of Nanoparticles by In Vivo Imaging

The traffic of 20 and 500 nm fluorescent polystyrene nanoparticles, aswell as VLP-Alexa488 was investigated in vivo using an UV light tool(LT-99D2-220, Lightools Research, CA) equipped with 470/40 nm excitationfilter and high resolution camera (KM_Dynax_SD). Mice were injected withthe fluorescent nanoparticles in the footpad as described in Example 7and pictures were taken at 2, 24 and 192 h after injection. In agreementwith flow cytometry data (Table 2), 2 h after injection VLP-Alexa488have reached the popliteal LN as detected by fluorescence microscopy.Similarly to VLP (30 nm), 20 nm nanoparticles (Table 3) were alsodetected, suggesting that small nanoparticles are able to drain in acell-free manner to the LN. On the contrary, at this early time point500 nm nanoparticles were not detected in the popliteal LN (Table 3),demonstrating a slower kinetics of traffic of larger particles to theLN. Flow cytometry analysis were performed 2 h after injection of 20 or500 nm nanoparticles. These results confirmed the findings with thelight tool imaging (Table 3). One possibility for the slower traffickingkinetics of 500 nm particles is that it takes place in part via DCuptake at the injection site and transport to the LN.

Twenty four hours after injection, 500 nm particles have reached thedraining LN as detected by fluorescence microscopy, although theassociated fluorescence was quantitatively less than in LN of miceinjected with 20 nm and VLP-Alexa488 nanoparticles. At later time points(192 h) VLP-Alexa488, 20 and 500 nm particles were still present at theinjection site and in the poplitcal LN, suggesting a depot formation andcontinuos delivery of antigen.

Taken together, these data suggest a size dependent mechanism of trafficof nanoparticles to the LN. Small particles, such as 20 nm andVLP-Alexa488 drain in a cell-free manner to LN at early time pointswhereas large panicles (500 nm) show slower kinetics due to requirementfor DC-mediated transport.

TABLE 3 Flow cytometry analysis of the trafficking of nanoparticles tothe popliteal LN 2 h after injection. The percentage of green cells inthe total LN cell gate is shown. Particle Size [nm] FL1 + LN cells 201.59 500 0.05* *the value is in the range of the background observed innaive animals

Example 10 DC-Dependence of the Delivery of Nanoparticles to the LN

Macrophages are thought to be non-migratory cells, therefore particlebearing macrophages are supposed to be LN-resident cells that hadacquired particles while in the LN. On the contrary, DC have thecapacity to engulf particles at the injection site and to migrate to thedraining LN. To investigate the relationship between the size of ananoparticle and the mechanism of trafficking to the LN, the fluorescentnanoparticles ranging in size from 20 to 2000 nm were injected in thefootpads of mice as described in Example 7, and the ratio between thenanoparticle-containing DC and macrophages was calculated (FIG. 5). Thelarger the injected particle (500-2000 nm),the more DCs are involved inits uptake in LN. Smaller particles (20-200 nm) were detected incomparable percentage of DC and macrophages, suggesting cell-freedraining and association with LN-resident APC.

In vivo imaging kinetic studies in wild type mice and in miceconditionally depleted of DCs (CD11c-DTR-GFP mice, Jung S. et al., 2002)showed that large particles (500 nm) reached the draining LN only in thepresence of DCs, wherease small (20 nm) nanoparticles traffickedefficiently also in DC-depleted animals. These data suggest that smallnanoparticles can target lymph node resideint DC populations. Indeed, asswown in Table 4, 20 nm particles and VLP (30 nm) associated with thelymph node-resident plasmacytoid DCs (PDCA-1⁺B220⁺), B cells(PDCA-1⁻B220⁺) and lymphoid DCs (CD11c⁺CD8⁺). Therefore, small particlesare useful to target LN-resident APC, whereas large particlesexclusively target DC at the injection site.

TABLE 4 Flow cytometry analysis of the trafficking of nanoparticles tothe popliteal LN 48 h after injection. The percentage of green cells inthe gate of the indicated LN cell populations is shown. Particle % FL1⁺% FL1⁺ % FL1⁺ B size [nm] Lymphoid DCs plasmacytoid DCs cells  20 12 8 430 (VLP) 13 16 2 1000 3 0 0

Example 11 Generation of Bone Marrow-Derived Dendritic Cells (BMDC) andTreatment with QβG10 batches

11 week old mice from the LPS-resistant (C3H/HeJ) and LPS-responding(C3H/HeN) strains were sacrificed and the femurs and tibias isolated andkept in PBS. After removing the flesh, bones were cut from both sidesand the bone marrow flushed out using a syringe filled with 10% FCSRPMI. Released cells were collected and passed through a 70 μm cellstrainer. Cells were washed and re-suspended at 20×10⁴ cells/ml in 10%FSC RPMI containing 10 ng/ml mouse GM-CSF (R&D). 2×10⁶ cells were platedin bacterial grade Petri dishes in 10 ml medium for 6 days. Thedifferentiation status of BMDC was ascertained on day 6 by analyzingcells for the expression of CD11c and CD11b by flow cytometry. At day 6of differentiation, BMDC were harvested from the Petri dishes, washedonce and plated in 96-well U-bottom plate (BD) at 5×10⁴/well. 6dilutions (4-fold) of QβG10 were prepared in a separate plate and addedto BMDC for 20 h.

ELISA for determination of IL-12: Microtiters plates (Maxisorp, Nunc)were coated overnight with 2 μg/ml rat anti-mouse IL-12 (p40/p70)monoclonal antibody (mAb, Pharmingen) in coating buffer (0.1M NaHCO₃, pH9.6). After washing (0.05% Tween 20/PBS) and blocking (2% BSA/0.05%Tween 20/PBS), 70 μl of supernatants from BMDC or two-fold dilutions ofrecombinant IL-12 (starting from 20 μg/ml) were added and incubated for2 h at room temperature (RT). After washing the plates, biotin-labeledrat anti-mouse IL-12 mAb (p40/p70, Pharmingen) was added at 1 μg/ml for1 h at RT. Plates were washed and incubated with 1 μg/mlstreptavidin-HRPO (Jackson laboratories) for 1 h at RT. After washing,o-phenylene diamine (OPD. Fluka) in citric acid buffer (pH 5) was addedfor 5 min and the reaction was stopped with 5% H₂SO₄. Optical density at490 nm was measured in an ELISA reader (Molecular Devices) and data wereanalyzed using the software Soft max Pro.

Results; Activation of BMDCs by Qβ packaged with G10 oligo is shown onFIG. 6. BMDCs were activated by QβG10 and hence secreted IL-12 in a dosedependent way (dose is given as equivalent of G10 oligonucleotidepackaged in the Qβ VLPs) while untreated control cells did not secreteany IL-12. Therefore G10 oligonucleotide packaged in Qβ VLPs is able toactivate BMDCs, demonstrating that the particles are taken up by thecells and oligonucleotide is made subsequently available for stimulationof the BMDCs.

Example 12 BMDC Release IL-12 upon AP205G10 Treatment

BMDC from C3H/HeJ mice were in vitro generated as described in Example11. Cells were plated in a 96-well plate and stimulated either mock orwith the indicated concentrations of AP205 reassembled with G10(AP205G10). Eighteen hours later the supernatants were collected andIL-12 was measured by a sandwich ELISA, as described in Example 11.AP205G10 induced a dose-dependent secretion of IL-12, while untreatedcells released IL-12 only to basal levels (Table 5). These resultsdemonstrate that AP205G10 is taken up by BMDC and activates them torelease IL-12.

TABLE 5 Dose response of AP205G10 induced IL-12 secretion by BMDCs.AP250G10 μg/ml IL-12 [pg/ml] 300 4699.7 150 4831.3 75 4535.9 37.5 3644.718.75 2760.3 9.375 1407.0 0 471.1

Example 13 Preparation and Characterization of Gelatin Nanoparticles asDelivery System for G10 Oligodesoxynucleotide (G10-ODN)

Preparation of plain Gelatin Nanoparticles (GNP) by two stepdesolvation: The procedure used was the original one described by(Coester et al. 2000) as follows. 1.25 g of gelatin type A (Bloom 175)was dissolved in 25 ml highly purified water (HPW) to 5% (w/w) undergentle heating to 50° C. A first desolvation step was initiated by theaddition of 25 ml acetone. After sedimentation of the precipitatedgelatin fractions for 60 seconds, the supernatant consisting ofdispersed gelatin as well as dissolved gelatin was discarded. Thesediment was dissolved again by the addition of 25 ml water underheating to 50° C. and the pH was adjusted to 2.5. In situ gelatinnanoparticles were formed during a second desolvation step by drop wiseaddition of 50 ml acetone under constant stirring (500 rpm). After 10min, 175 μl of glutaraldehyde (25%) were added to the reaction vessel tocrosslink the nanoparticles. Finally, after stirring for 12 hours in anextractor hood, the particles were purified by three-fold centrifugation(20000 g for 20 min) and redispersion in acetone/water (30/70), the laststep in HPW alone. The purified nanoparticles were stored as dispersionin HPW (conductivity <0.04 μs/cm) at 4-8° C. The following standardprocess parameters are critical for nanoparticle preparation: (a)Temperature before the first and second desolvation step: 50° C.; (b)stirring speed: 500-700 rpm; (c) precipitation time after the firstdesolvation step: 60 sec; (d) speed of acetone addition (seconddesolvation step): 3-5 ml/min.

Determination of nanoparticle size: Particle sizes were determinedemploying dynamic light scattering (DLS) technology. DLS experimentswere performed with a Zetasizer ZS (Malvern Instruments, Worcestershire,England) using NIBS™-technology (Non Invasive Back Scattering) at astatic detection angle of 173°. The nanoparticles were diluted insterile filtered, highly purified water and measured in concentrationsbetween 30 and 100 μg/ml. Due to these low concentrations, thenanoparticles did not influence the viscosity of the dispersion, so thatthe viscosity of pure water which is of 0.8872 cP at 25° C. was used.The experiments were performed at 25° C.

Cationisation of plain Gelatin Nanoparticles: A new, modified version ofthe original method described by Coester (2003) using cholaminechloridehydrochloride was used (Ahlers et al. 2005; Coester 2003; Zwiorek et al.2004). An aqueous dispersion of plain nanoparticles was adjusted to pH4.5 and a molar excess of the cationization agent cholamine chloridehydrochloride (e.g. 50 mg per 500 mg nanoparticles) was added underconstant stirring. After 5 minutes of incubation, 50 mg of1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (EDC) wasadded to activate the free carboxyl groups on the particles to reactwith cholaminechloride hydrochloride. During the cationization reactionthe primary amino groups of the cholaminechloride hydrochloride canreact with two possible functional groups on the nanoparticle's surface.The first interaction sites are residual aldehyde groups derived fromthe only mono-functionally bound cross linking reagent glutaraldehyde.Furthermore, the primary amino groups can react with activated carboxylgroups on the nanoparticles surface. The reaction was stopped after 1 hand the nanoparticles were purified by 3-fold centrifugation andredispersion. analogous to the purification of plain nanoparticles. Thefinal particles were characterized by size determination using dynamiclight scattering, and measurement of the Zeta potential. Each assignedsize and corresponding polydispersity index was the mean of 10 subruns.The Zeta potential was determined using the same instrument andcalculated as the mean of 10 individual measurements in PBS. Particlesshowing a positive charge of 5 mV or higher in PBS were used forloadings.

G10-ODN loading on cationized gelatin nanoparticles: G10-ODN wasemployed either in an aggregated or non-aggregated form (omitting theaggregation step in the protocol for ODN G10 preparation, Example 2) inpurified water and in PBS. For the cellular assays described bewlo thecontents were 1% for GNP and 0.1% for G10-ODN leading to a targetedloading of 10% (w/w) in PBS. For the mouse model study described below,a G10-ODN content of 0.25% and GNP of 2.5% were prepared as wellproviding a 10% (w/w) loading in PBS. The loading experiment isexemplary described for a targeted (aspired) 10% (w/w) G10-ODN loadingon GNP in PBS, and the same procedure was adapted for the other targetedloading percentages using aggregated or non-aggregated G10-ODN. 43.3 μlof an aqueous GNP dispersion containing 1.2 mg GNP were transferred toan Eppendorf™ 1.5 ml cap under aseptic conditions. Then 1086 μl of PBSwere added and mixed at the same time with the pipette. In the nextstep, 71.4 μl of 010 stock solution containing 120 μg of aggregated G10,were added into the dispersion by quick but gentle up and down pipettingfor at least 20 seconds. The prepared sample was subsequently incubatedfor 1 h 15 min at 22° C. and 750 rpm on a Thermomixer™ device.

Determination of G10-ODN loading efficiency (Zillies and Coester 2004):After incubation, the G10-ODN loaded GNP samples to be tested forsuccessful loading were centrifuged for 1 h at 15000 g. Aftercentrifugation the supernatant was carefully separated from theremaining pellet and analyzed spectrophotometrically at a wavelength of260 nm. A reference of unloaded GNP of the same concentration (1 mg/ml)and a reference of G10-ODN of the same concentration (100 μg/ml) wereprepared and centrifuged along. G10-ODN loading in percent wascalculated from the percentage free G10-ODN, which was obtained bysubtracting the measured absorption of the particle reference from thesample result and then dividing this result by the measured absorptionof the G10-ODN reference, and multiplying by hundred. Finally, theloading efficiency was calculated by subtracting the percentage freeG10-ODN from 100%.

Results of G10-ODN loading determination: Results of loading efficiencywere obtained by various GNP batches. However, the preparation processand especially cationization was the same in each case. Hence, loadingscan be compared as well as among batches with different particle sizes.All results given in percent are mass percentage (w/w) calculated on theemployed masses of G10-ODN and GNP. As shown in Tables I-III fornon-aggregated G10-ODN and Tables IV-V for aggregated G10-ODN, G10-GNPswith loading percentage from 3-15% (non-aggregated G10-ODN) or 5-10%(aggregated G10-ODN) were successfully obtained. Optimal loading mediumwas PBS, where no flocculation was detected in the preparations. Resultsof not aggregated G10-ODN show sufficient bindings over all with atendency to decreased efficiency from 5 to 10% loading in water andwithout this tendency in PBS. The loading of 15% G10-ODN onto GNP showsonly a slightly lower efficiency.

TABLES I AND II loading efficiency [%] of not aggregated G10 on GNP (314nm) in water (left) and GNP (218 nm) in PBS (right). The single numberson top of each sub table give the targeted G10-ODN loading onto GNP inpercent. 3 3 particle reference 0.132 (n = 3, S.D. = 0.0021) particlereference 0.103 (n = 3, S.D. = 0.006) ODN reference 3% 0.404 (n = 3,S.D. = 0.0029) ODN reference 3% 0.904 sample G10 3% (water) 0.117 (n =3, S.D. = 0.0006) sample G10 3% (PBS) 0.127 (n = 3, S.D. = 0.001)loading [%] 100.000 loading [%] 97.476 5 5 particle reference 0.132 (n =3, S.D. = 0.0021) particle reference 0.103 (n = 3, S.D. = 0.006) ODNreference 5% 0.652 (n = 3, S.D. = 0.0021) ODN reference 5% 1.647 sampleG10 5% (water) 0.132 (n = 3, S.D. = 0.021) sample G10 5% (PBS) 0.106 (n= 3, S.D. = 0.0066) loading [%] 100.000 loading [%] 99.798 8 8 particlereference 0.132 (n = 3, S.D. = 0.0021) particle reference 0.103 (n = 3,S.D. = 0.006) ODN reference 8% 1.079 (n = 3, S.D. = 0.0026) ODNreference 8% 2.673 sample G10 8% (water) 0.149 (n = 3, S.D. = 0.0000)sample G10 8% (PBS) 0.100 (n = 3, S.D. = 0.0107) loading [%] 97.888loading [%] 100.000 10 10 particle reference 0.132 (n = 3, S.D. =0.0021) particle reference 0.103 (n = 3, S.D. = 0.006) ODN reference 10%1.344 ODN reference 10% 3.356 sample G10 10% (water) 0.186 (n = 3, S.D.= 0.0010) sample G10 10% (PBS) 0.166 (n = 3, S.D. = 0.0079) loading [%]95.700 loading [%] 98.113

TABLE III loading efficiency [%] of not aggregated G10 onto GNP (218 nm)in PBS particle reference 0.105 (n = 3, S.D. = 0.0006) ODN reference 15% 2.557 sample G10 15% (PBS) 0.238 (n = 3, S.D. = 0.0154) loading [%]94.798

TABLE IV loading efficiency [%] of aggregated G10 onto GNP (218 nm) inPBS ODN-reference 0.827 (n = 5, S.D. = 0.002) particle reference 0.145(n = 5, S.D. = 0.004) sample G10 5% aggl 0.176 (n = 5, S.D. = 0.005)loading [%] 96.251 (n = 5, S.D. = 1.089) ODN-reference 0.827 (n = 4, SD.= 0.002) particle reference 0.145 (n = 4, S.D. = 0.004) sample G10 10%aggl 0.159 (n = 4, S.D. = 0.001) loading [%] 98.277 (n = 4, S.D. =0.425)

TABLE V Loading efficiency [%] of aggregated G10 onto GNP (218 nm) inHPW particle reference 0.106 (n = 3, S.D. = 0.0005) ODN reference 1.870(n = 3, S.D. = 0.0037 sample G10 10% (water) 0.285 (n = 3, S.D. =0.0034) loading [%] 90.426

G10-loaded gelatin nanoparticles (GNP-G10) induce IL-12 secretion fromBMDC: GNP-G10 were prepared as described above. To evaluate the effectof unbound G10, an aliquot of GNP-G10 preparation was centrifuged at16000 g for 1 h at 4° C. and the supernatant was collected (supGNP-G10).BMDC from C3H/HeJ mice were generated in vitro, as described in Example11. Cells were plated in a 96-well plate and stimulated either mock orwith the indicated concentrations of GNP-G10 or supGNP-G10 (given astheir molar concentration of G10). Eighteen hours later the cellsupernatants were collected and IL-12 was measured by a sandwich ELISA,as described in Example 11. GNP-G10 induced secretion of IL-12 from BMDCat all tested concentrations (Table VI). In contrast, BMDC did notrelease significant amounts of IL-12 upon treatment with supGNP-G10.These results suggest that GNP-G10 are taken up by BMDC and triggerrelease of IL-12. In addition, these data show that G10 is efficientlyloaded on GNP, as supGNP-G10 did not induce IL-12 secretion from BMDC.GNPs without G10 do not induce IL-12 secretion in mouse dendritic cells.

TABLE VI GNP-G10 induced secretion of IL-12 from BMDC. IL-12 [pg/ml] G10[nM] GNP-G10 std supGNP-G10 std 5200 7841.0 846.5 382.7 42.4 2600 7444.4365.8 232.9 10.8 1300 7382.9 379.3 83.3 1.4 650 6968.6 271.9 58.2 13.7325 7701.7 210.6 371.2 456.2 162.5 7007.7 348.7 52.0 3.7 0 41.3 21.541.3 21.5

G10-loaded gelatin nanoparticles (GNP-G10) are protective in an mousemodel of asthma: An experimental asthma model of allergic airway (HesselE M et al., J. Exp. Med. 2005, 202, 1563) is used to assess the effectsof GNP-G10 on total IgE level. For sensitization, four groups of mice(groups B-E, five mice per group) are injected intraperitonealy (i.p.)with 150 μg ragweed (Ambrosia aremisiifolia) pollen extract (RW, Greer)mixed with 450 μg of Alum (Alhydrogel 2.0% Brenntag Biosector, Denmark)on day 0 and 3. Mice from one group (group A) are treated i.p. with PBSonly. On day 10, mice from group C are injected subcutaneously with 300μg QbG10 (equivalent to 60 μg G10), mice from group D with 600 μgGNP-G10 (equivalent to 60 μg G10), mice from group E with 600 μg GNP-G10aggregated (equivalent to 60 μg G10) mice from groups A and B with PBS.Blood is sampled from all animals prior to injection. On day 17, allanimals are bled, and total IgE titer is determined. The total IgE levelin blood is measured by ELISA. Briefly, an ELISA plates (96 wellMAXIsorp, NUNC immuno plate #442404) is coated with anti-IgE (BDPharmingen, #553413) at a concentration of 5 μg/ml in coating buffer(0.1M NaHCO₃, pH 9.6) over night at 4° C. The plates are then washedwith wash buffer (PBS/0.05% Tween) and blocked for 2 h at 37° C. with 2%BSA in wash buffer. The plate is then extensively washed and thenincubated with 1 to 20 diluted mouse sera or serially diluted mouse IgEstandard (BD Pharmingen, #557079). The plate is incubated at RT for 2 hand then extensively washed with wash buffer. Bound specific mouseantibodies are then detected by one hour incubation with aHRPO-labelled, epsilon chain specific, rat anti-mouse IgE antibody(Southern Biotech #H021-NBB4C). After extensive washing with washbuffer, plates are developed with OPD solution (1 OPD tablet, 25 ml OPDbuffer and 8 μl H₂O₂) for 6 minutes and the reaction is stopped with 5%H₂SO₄ solution. Plates are then read at OD 450 nm on an ELISA reader(Biorad Benchmark). The concentrations of IgE in sera is calculated withGraphPad Prism4 using the mouse IgE standard as reference.

REFERENCES FOR EXAMPLE 13

-   Ahlers, Michael, et al. Biodegradable gelatin nanoparticles and    procedure for their production. (Deutsche Gelatine-Fabriken Stoess    A.-G., Germany, assignee. Patent 102004041340, 20060223. (2005).-   Coester, C. J., et al. “Gelatin nanoparticles by two step    desolvation-a new preparation method, surface modifications and cell    uptake.” Journal of Microencapsulation 17.2 (2000): 187-93.-   Coester, Conrad. Development of a new carrier system for    oligonucleotides and plasmids based on gelatin nanoparticles. New    Drugs [1], 14-17, 2003.-   Zillies, Jan and Coester, Conrad. Evaluating gelatin based    nanoparticles as a carrier system for double stranded    oligonucleotides. Journal of Pharmacy & Pharmaceutical Sciences    7[4], 17-21, 2004.-   Zwiorek, Klaus, Kloeckner, Julia, Wagner, Ernst, and Coester,    Conrad. Gelatin nanoparticles as a new and simple gene delivery    system. Journal of Pharmacy & Pharmaceutical Sciences 7 [4], 22-28,    2004.

Example 14 Detection of IFN-Alpha Production in Cells by ELISA

Human peripheral blood mononuclear cells (PBMC) were isolated from buffycoats and treated with immunostimulatory nucleic acids in RPMI mediumcontaining 10% fetal calf serum (FCS) for 18 h. IFN-alpha in thesupernatants was measured by sandwich ELISA, using an antibody setprovided by PBL Biomedical Laboratories. Briefly, ELISA plates werecoated with 5 μg/ml of the capture anti-IFN-alpha antibody. Afterwashing, plates were blocked with 0.5% BSA for 1 h at room temperature(RT). Supernatants from treated and control PBMC, as well as recombinantIFN-alpha were added to the plate and incubated for 2 h at 37° C. Rabbitpolyclonal anti-IFN-alpha serum was used as detection antibody andincubated for 1 h at RT. Peroxidase-conjugated goat anti-rabbit serumwas subsequently added to the plates for 1 h at RT. Washing step wasperformed after each incubation. The color reaction was developed usingthe enzyme substrate o-phenylendiamine and read at 490 nm in an ELISAreader device. The data are expressed as pg/ml secreted IFN-alphaaccording to a standard curve of recombinant IFN-alpha.

Example 15 Flow Cytometry Analysis Using Fluorochrom-ConjugatedAntibodies

PBMC are stimulated for 3-6 h with immunostimulatory nucleic acidsfollowed by incubation with brefeldin A for additional 4 h. Cells arethen stained with DC-specific surface markers, permeabilized andintracellularly stained for IFN-alpha.

Example 16 Effects of QβG10 on Eosinophilla after Sensitization withRagweed Pollen and One Challenge on Day 11

An experimental asthma model of allergic airway (Hessel E M et al., J.Exp. Med. 2005, 202, 1563) was used to assess the effects of QβG10 oneosinophilia, a pathologic maker of asthma. For sensitization, threegroups of mice (five mice per group) were injected intraperitonealy(i.p.) with 150 μg ragweed (Ambrosia artemisiifolia) pollen extract (RW,Greer) mixed with 450 μg of Alum (Alhydrogel 2.0% Brenntag Biosector,Denmark) on day 0 and 3. On day 9, mice from group B received 375 μg ofQβG10 subcutaneously (s.c.). On day 10, mice from group C received 375μg of QβG10 subcutaneously. Mice from of group A received PBS ascontrol. On day 11, all mice were challenged with 200 μg of RW in PBSintranasally (i.n.). 48 hours after the challenge mice were sacrificedand the lungs were washed with PBS (Table 6). The cells contained in thebroncho alveolar lavage (BAL) were analysed by FACS. Eosinophils wereidentified by high CCR3 (Mouse CCR3 Phycoerythrin MAb, R & D)expression. As shown in Table 7, the total amount of cells in BAL wasstrongly reduced in QβG10 treated compared to PBS control mice. Thereduction was more pronounced when QβG10 was given 48 hours (61%,p<0.05) before the challenge than 24 hours (52%) before the challenge.As shown in Table 8, the amount of eosinophils was strongly reduced inQβG10 treated compared to PBS control mice. Again, the reduction wasmore pronounced when QβG10 was given 48 hours (70%, P<0.05) than 24hours (60%, p<0.05) before the challenge. Thus, QβG10 treatment preventsthe development of asthma in a murine model for asthma.

TABLE 6 Experimental Protocol. Day 0 Day 3 Day 9 Day 10 Day 11 Day 13 ARW RW PBS s.c. RW Analysis alum alum i.p. i.p. B RW RW QβG10 RW Analysisalum alum s.c. i.p. i.p. C RW RW QβG10 RW Analysis alum alum s.c. i.p.i.p.

TABLE 7 Total Cell Counts in BAL. Group A Group B Group C mouse 1210′000 60′000 180′000 mouse 2 636′000 66′000 312′000 mouse 3 330′00060′000 120′000 mouse 4 246′000 366′000 102′000 mouse 5 480′000 360′00036′000 average 380′400 182′400 150′000 reduction 52% 61%

TABLE 8 Eosinophil counts in BAL. Group A Group B Group C mouse 1 61′69611′208 19′855 mouse 2 90′166 15′913 32′487 mouse 3 106′682 9′811 12′786mouse 4 36′300 64′255 13′276 mouse 5 82′757 58′752 4′222 average 75′52031′988 16′525 reduction 63% 70%

Example 17

Effects of QβG10 on Eosinophilla after Sensitization with Ragweed Pollenand Two Challenges (Day 11 and 15)

A stronger experimental asthma model of allergic airway (modified fromHessel E M et al., J. Exp. Med. 2005, 202, 1563) was also used to assessthe effects of QβG10 on eosinophilia and IgE concentration in blood. Forsensitization, two groups of mice (five mice per group) were injectedintraperitonealy with 150 μg RW pollen extract in 450 μg of alum on day0 and 3. On day 9 and 13, mice from group B were subcutaneously injectedwith 375 μg of QβG10. Mice from of group A received PBS as control. Onday 11 and 15, mice were challenged with 200 μg of RW in PBSintranasally. 48 hours after the challenge mice were sacrificed and thelungs were washed with PBS (Table 9). The cells contained in the BALwere analyzed by FACS as described in Example 16. As shown in Table 10and 11, the total amount of cells (51%, p<0.01) and the amount ofeosinophils (62%, p<0.001) were strongly reduced in QβG10 treatedcompared to PBS control mice. Thus, QβG10 prevents the development ofasthma in a stronger murine model for asthma.

The total IgE level in blood was measured by ELISA. Briefly, an ELISAplates (96 well MAXIsorp, NUNC immuno plate #442404) were coated withanti-IgE (BD Pharmingen, #553413) at a concentration of 5 μg/ml incoating buffer (0.1M NaHCO3, pH 9.6) over night at 4° C. The plates werethen washed with wash buffer (PBS/0.05% Tween) and blocked for 2 h at37° C. with 2% BSA in wash buffer. The plate was then extensively washedand then incubated with 1 to 20 diluted mouse sera or serially dilutedmouse IgE standard (BD Pharmingen, #557079). The plate was incubated atRT for 2 h and then extensively washed with wash buffer. Bound specificmouse antibodies were then detected by one hour incubation with aHRPO-labelled, epsilon chain specific, rat anti-mouse IgE antibody(Southern Biotech #H021-NBB4C). After extensive washing with washbuffer, plates were developed with OPD solution (1 OPD tablet, 25 ml OPDbuffer and 8 μl H₂O₂) for 6 minutes and the reaction was stopped with 5%H₂SO₄ solution. Plates were then read at OD 450 nm on an ELISA reader(Biorad Benchmark). The concentrations of IgE in sera were calculatedwith GraphPad Prism4 according to the standard. As shown in Table 12,the total IgE level in mice treated with QβG10 are significantly(P<0.05) reduced comparing to untreated mice.

TABLE 9 Experimental Protocol. Day 0 Day 3 Day 9 Day 11 Day 13 Day 15 D17 A RW RW PBS RW PBS RW Analysis alum alum i.p. i.p. B RW RW QβG10 RWQβG10 RW Analysis alum alum s.c. s.c. i.p. i.p.

TABLE 10 Total Cell Counts in BAL. Group A Group B mouse 1   877′250437′250 mouse 2 ND 805′750 mouse 3 1′057′375 534′875 mouse 4 1′199′000460′625 mouse 5 1′287′000 496′375 average 1′105′156 546′975 reduction51%

TABLE 11 Eosinophil Counts in BAL. Group A Group B mouse 1 611132 208045mouse 2 ND 492788 mouse 3 802727 337733 mouse 4 901931 244154 mouse 5949650 276519 average 816360 311848 reduction 62% ND: not determined

TABLE 12 IgE Concentration in sera (μg/ml). Group A Group B mouse 1 4.60.7 mouse 2 0.7 0.4 mouse 3 3.5 0.4 mouse 4 4.6 0.6 mouse 5 1.7 0.3average 3.0 0.5 reduction 84.5%

Example 18 Duration of the Effect of QβG10 and Interaction of QβG10 withRW Antigen

Mice were sensitized with RW mixed with alum as in Example 16 on day 0and 3. On day 10 and day 12, mice received QβG10, QβG10 mixed with RW(20 μg) or PBS subcutaneously. 6 and 14 days after the treatment, micewere challenged with RW. 48 hours after the challenge, IgE level in serawas analyzed (Table 13). The data clearly show that IgE level in QβG10or QβG10 mixed with RW treated mice is significantly reduced comparingto PBS treated controls (Table 14) and no clear difference has been seenbetween QβG10 and QβG10 mixed with RW.

TABLE 13 Experimental Protocol. Day 0 Day 3 Day 10 Day 12 Day 19 Day 21Day 26 Day 29 Day 40 Day 42 A RW RW PBS s.c. PBS s.c. RW i.n. Analysisalum alum i.p. i.p. B RW RW QβG10 QβG10 RW i.n. Analysis alum alum s.c.s.c. i.p. i.p. C RW RW QβG10 QβG10 RW i.n. Analysis alum alum RW s.c. RWs.c. i.p. i.p. D RW RW PBS s.c. PBS s.c. RW i.n. Analysis alum alum i.p.i.p. E RW RW QβG10 QβG10 RW i.n. Analysis alum alum s.c. s.c. i.p. i.p.F RW RW QβG10 QβG10 RW i.n. Analysis alum alum RW s.c. RW s.c. i.p. i.p.

TABLE 14 IgE Concentration in sera (μg/ml). A B C D E F mouse 1 1.620.13 0.30 1.70 0.47 0.30 mouse 2 0.78 0.16 0.37 0.62 0.05 0.33 mouse 30.56 0.10 0.21 0.70 0.16 0.31 mouse 4 1.98 0.22 0.12 1.47 0.42 0.29mouse 5 0.54 0.24 0.22 0.60 0.35 0.29 average 1.10 0.17 0.24 1.02 0.290.30 reduction 84.5% 77.8% 71.5% 70.2%

Example 19 Specificity of QβG10

Mice are sensitized with RW and Fel d1 (Cytos, Switzerland) on day 0 and3. On day 10. mice are treated with either PBS (group A and B), or QβG10(group C and D), or QβG10 mixed with RW (group E and F) or QβG10 mixedwith Fel d1. On day 12, mice from group A, C, E and G are challengedwith RW mixed with alum. On the other hand, mice from group B, D, F andH are challenged with Fel d1. On day 14, all mice receive the sametreatment as on day 10. On day 16 all the mice are challenged as on day12. 48 hours after the last challenge, RW and Fel d1 specific IgE in theBAL and blood are measured and infiltrating cells in BAL are analyzed(Table 15).

TABLE 15 Experimental Protocol. Day 0 Day 3 Day 10 Day 12 Day 14 Day 16Day 18 A RW Feld1 RW Feld1 PBS s.c. RW i.n. PBS s.c. RW i.n. Analysisalum i.p. alum i.p. B RW Feld1 RW Feld1 PBS s.c. Feld1 i.n. PBS s.c.Feld1 i.n. Analysis alum i.p. alum i.p. C RW Feld1 RW Feld1 QβG10 RWi.n. QβG10 RW i.n. Analysis alum i.p. alum i.p. s.c. s.c. D RW Feld1 RWFeld1 QβG10 Feld1 i.n. QβG10 Feld1 i.n. Analysis alum i.p. alum i.p.s.c. s.c. E RW Feld1 RW Feld1 QβG10 RW i.n. QβG10 RW i.n. Analysis alumi.p. alum i.p. RW s.c. RW s.c. F RW Feld1 RW Feld1 QβG10 Feld1 i.n.QβG10 Feld1 i.n. Analysis alum i.p. alum i.p. RW s.c. RW s.c. G RW Feld1RW Feld1 QβG10 RW i.n. QβG10 RW i.n. Analysis alum i.p. alum i.p. Feld1s.c. Feld1 s.c. H RW Feld1 RW Feld1 QβG10 Feld1 i.n. QβG10 Feld1 i.n.Analysis alum i.p. alum i.p. Feld1 s.c. Feld1 s.c.

Example 20 Comparison G10 and QβG10

The effects of G10 and QβG10 were compared in the ragweed allergicmodel. Three groups of mice were sensitized with ragweed on day 0 andday 3 by i.p. injection of ragweed mixed with alum. On day 10 and 12,mice from group B and C were treated with G10 or QβG10 respectively.Mice from group A received PBS as control. All mice were challenged with200 μg of RW by i.n. administration and two days after the challengemice were sacrificed. Infiltrating cells and IL-4 concentration in BALand IgE level in blood were analysed (Table 16).

As shown in Table 17, whereas the total amount of cells in BAL was notsignificantly changed in G10 treated mice, it was strongly reduced inQβG10 treated mice compared to PBS control mice (32%, p<0.05).Similarly, the amount of eosinophils was strongly reduced (64.1%,p<0.01) in QβG10 treated compared to PBS control mice and the changes inG10 treated mice were not significant (Table 18). Thus, whereasnon-packaged G10 had not significant effects on infiltrating cells inBAL, G10 packaged in Qβ prevented cells infiltrating to the lung. Asshown in Table 19, whereas the changes of IgE level in mice treated withG10 were not significant, the QβG10 treated mice had significantly(p<0.001) reduced blood IgE levels in comparison with PBS treated mice.As shown in Table 20, whereas the changes of IL4 level in BAL from micetreated with G10 were not significant, the QβG10 treated mice hadsignificantly (p<0.001) reduced BAL IL-4 levels in comparison with PBStreated mice. Therefore, when G10 was packaged in Qβ, it reduced thepathological markers of asthma and allergy.

TABLE 16 Experimental design. Day 0 Day 3 Day 10 Day 12 Day 14 Day 16 ARW RW PBS s.c. PBS s.c. RW i.n. Analysis alum alum i.p. i.p. B RW RW G10s.c. G10 s.c. RW i.n. Analysis alum alum i.p. i.p. C RW RW QβG10 QβG10RW i.n. Analysis alum alum s.c. s.c. i.p. i.p.

TABLE 17 Total Cell Counts in BAL and changes relative to group A. GroupA Group B Group C mouse 1 402000 439500 274500 mouse 2 358500 325500319500 mouse 3 309000 357000 112500 mouse 4 306000 196500 274500 mouse 5337500 279000 177000 average 342600 319500 231600 % reduction 7 32

TABLE l8 Eosinophils in BAL and changes relative to group A. Group AGroup B Group C mouse 1 158020 146095 37134 mouse 2 109612 76898 65099mouse 3 85261 95275 20344 mouse 4 72211 43605 33118 mouse 5 97900 5371631945 average 104601 83118 37528 % reduction 20.5 64.1

TABLE 19 IgE Concentration (μg/ml) and changes relative to group A GroupA Group B Group C mouse 1 0.93 0.49 0.16 mouse 2 0.86 0.30 0.11 mouse 30.82 0.66 0.17 mouse 4 0.44 0.52 0.18 mouse 5 0.54 0.49 0.19 average0.72 0.49 0.16 % reduction 31.6 77.6

TABLE 20 IL-4 Concentration (pg/ml) in BAL and changes relative to groupA. Group A Group B Group C mouse 1 17.7 24.9 1.4 mouse 2 26.2 25.0 1.6mouse 3 51.2 35.9 1.5 mouse 4 37.6 6.5 4.3 mouse 5 49.1 4.9 2.4 Average36.4 19.4 2.2 % reduction 46.5 93.8

Example 21

Treatment of Cat Allergy with QβG10

Mice are sensitized by intraperitoneal injection with 5 μg of Fel d1 onday 1 and 14, and then boosted intranasally with 1 μg of Fel d1 on days28, 29, 30 and 33. Mice are injected subcutaneously with 375 μg of QβG10or PBS on days 37, 39 and 41. On day 43, mice are challengedintranasally with 1 μg of Fel d1 and the core temperature of the mice ismeasured rectally using a rectal probe digital thermometer. Mast celldegranulation is monitored in the cat allergy model. Mice are injectedsubcutaneously with 375 μg of QβG10 on day 0 and day 3. On day 5, miceare injected intradermally with 50 μl of 1:5 diluted cat allergic seraor purified IgE from that serum or control serum. 4 or 24 h later, miceare injected intravenously with 10 μg of Fel d1 plus Evans blue dye. Themice are sacrificed 30 min after the intravenous challenge and the bluestaining reaction is analyzed. QβG10 treated mice have reduced bluestaining as compared to untreated mice.

Example 22 Treatment of Bee Venom Allergy with QβG10

Female 6 to 8 week-old CBA/J mice are obtained from Harlan (Zeist, TheNetherlands). Animals are sensitized every other week by s.c. injectionin the ventral region and at the base of the tail of 0.1 μg PLA2adsorbed to 1 mg alum. Two protocols are applied for QβG10 therapy.First, presensitized mice receive daily s.c. injections of 375 μg ofQβG10 for 6 consecutive days. IgE levels are measured 48 hours after thelast QβG10 injection. Second, naive mice are injected s.c. with 375 μgof QβG10 on day 0 and day 3. Two weeks after the last QβG10 injection,mice are sensitized with PLA2 in alum. IgE levels are measured 7 daysafter PLA2 sensitization. Control groups receive either PBS or 30 μgnative PLA2. For the induction of anaphylactic responses, animals areinjected i.p. with 30 μg native PLA2 in PBS, and rectal temperature ismonitored with a calibrated digital thermometer. In comparison tountreated mice, the temperature drop in QβG10 treated mice is lesspronounced.

Example 23 Treatment of House Dust Mite Allergy with QβG10

Male CBA/CaH mice are sensitized with an intraperitoneal injection of 5μg of Der P1+1 mg of alhydrogel. For intranasal instillations, 25 μlaliquots containing 5 μg of Der P1 or sterile saline are applied to thenoses of anesthetized mice. Two protocols are applied QβG10 therapy.First, presensitized mice receive daily s.c. injections of 375 μg ofQβG10 for 6 consecutive days in. IgE levels are measured 48 hours afterthe last QβG10 injection. Second, naive mice are injected s.c. with 375μg of QβG10 on day 0 and day 3. Two weeks after the last QβG10injection, mice are sensitized with Der P1 in alum. IgE levels aremeasured 7 days after Der P1 sensitization. QβG10 treated mice exhibitlower IgE concentrations than the untreated control.

Example 24 Treatment of a Patient Suffering from Atopic Dermatitis withQβG10 and Assessment of Efficacy

24.1 Assessment of Symptoms of Atopic dermatitis: Total body involvementis estimated with the use of shapes of 100 to 1000 cm² or by the rule ofnine which assigns standard measurements to body parts on the basis ofthe size of the body part. The total body score is the sum of theindividual scores, on a scale of 0 to 3, for erythema, edema, pruritus,oozing crusting, excoriation, and lichenification of all involved skin,dryness of noninvolved skin, and sleep loss. The investigator gradesaffected skin areas of the test persons on a scale of 0 to 3 for theseverity of erythema, edema, oozing or crustating, excoriation, andlichenification of involved skin and dryness of noninvolved skin. Thetest persons grade the pruritus of the selected areas on a 10-cm visualanalogue scale, with severe at the bottom and absent at the top; thisgrade is converted to a score of 0 to 3 for analysis. To ensureconsistency in the assessment of efficacy, all the investigators receivea manual and participate in a on-day, centralized training course, aswell as on-site training.

24.2 Treatment with QβG10: A group of test persons suffering from Atopicdermatitis is treated at least three times in intervals of 1 week with300 μg QβG10 (Qβ VLP packaged with G10 (SEQ ID NO:27)), a control groupwith comparable symptoms is treated with placebo. Dependent on theaverage symptom score of the test group the treatment of the test groupcan be repeated up to a total of 6 to 10 vaccinations with 300 μg QβG10each, the control group is always treated with placebo in parallel.

24.3 Assessment of Efficacy: Total body involvement is assessed beforethe first treatment and at the end of the study. The symptoms onselected skin areas individually chosen for each patient are assesseddirectly before each treatment and in intervals up to 3 months after thelast treatment. The changes in the symptom scores of the test group atthe beginning and at the end of the study are compared to that of thecontrol group.

Example 25 Efficacy Parameters Atopic Dermatitis

The Physician's Global Assessment (PGA) of Atopic Dermatitis (AD): Theinvestigator will rate the overall disease severity using the scaledescribed in Table 21. The rating is to be assigned in consideration ofthe patient's current condition, without reference to previousassessments.

Eczema Area and Severity Index (EASI): The extent and severity ofdermatitis over 4 body surface areas will be calculated using the EczemaArea and Severity Index (EASI) (Hanifin J. M. et al. (2001), Exp.Dermatol. 10:11-18). The four body regions are assigned proportionatebody surface areas (BSA) such that the head and neck together comprise10% BSA, the front and back of the trunk each comprise 15% BSA, each armcomprises 10% BSA, and each leg comprises 20% BSA. The percentage ofarea involved for each of the body regions is given a score, rangingfrom 0 to 6, where 0=no eruption, 1=<10%, 2=10% to 29%, 3=30% to 49%,4=50% to 69%, 5=70% to 89%, and 6=90% to 100%. The four key symptoms ofatopic dermatitis to be assessed are: Erythema, Induration/papulation,Excoriation (linear abrasions of the skin due to scratching), andLichenification. Each of these symptoms will be scored at each of thefour body regions using a scale of 0 to 3, with half-steps allowed,where 0=none, 1=mild, 2=moderate, and 3=severe. The definitions outlinedin Table 22 will be used.

The score for each body region is obtained by multiplying the sum of theseverity scores of the four key symptoms by the percentage of areainvolved on the body region, then multiplying the result by the constantweighted value assigned to the particular body region (see Table 23).The EASI score is the sum of the four body region scores.

Patient's Assessment of Pruritus Score: At each study visit, the patientwill be asked to assess the severity of pruritus since the last visitusing the scale shown in Table 24.

TABLE 21 Physician's Global Assessment (PGA) of Atopic Dermatitis. ScoreGrade Definition 0 Clear No inflammatory signs of atopic dermatitis 1Almost clear Just perceptible erythema, and just perceptiblepapulation/induration 2 Mild disease Mild erythema, and mildpapulation/induration 3 Moderate disease Moderate erythema, and moderatepapulation/ induration 4 Severe disease Severe erythema, and severepapulation/ induration 5 Very severe disease Severe erythema, and severepapulation/ induration with oozing or crusting

TABLE 22 Severity scoring of key symptoms. Induration/ ErythemaPapulation Excoriation Lichenification None No evidence of Noperceptible No evidence of excori- No evidence of skin thicken- (0)erythema elevation ations ing Mild Very light pink, Barely perceptibleScant evidence of exco- Slight thickening of the skin, (1) faintlydetectable elevation riations with no sign of discernible only by touchand erythema deeper skin damage (ie, with markings minimally erosion,crust) exaggerated Moderate Dull red, clearly Clearly perceptible,Several linear marks of Definite thickening of the (2) distinguishablebut not extensive, skin, with some showing skin, with skin markingserythema elevation evidence of deeper skin exaggerated so that they forminjury (ie, erosion, crust) a visible criss-cross pattern Severe Deep,dark red Marked and exten- Many erosive or crusty Thickening induratedskin (3) sive elevation lesions with skin markings visibly portraying anexaggerated criss-cross pattern

TABLE 23 Eczema Area and Severity (EASI) Calculation (patients of 8years and older). Body region EASI Score^(a,b) Head/neck (E + IP + Ex +L) × Area × 0.1 Upper limbs (E + IP + Ex + L) × Area × 0.2 Trunk (E +IP + Ex + L) × Area × 0.3 Lower limbs (E + IP + Ex + L) × Area × 0.4EASI = Sum of the above 4 body region scores ^(a)Symptoms: E = erythema,IP = induration/papulation, Ex = excoriation, L = lichenification.^(b)Area: 0 = no eruption, 1 = <10%, 2 = 10%-29%, 3 = 30%-49%, 4 =50%-69%, 5 = 70%-89%, and 6 = 90%-100%.

TABLE 24 Patient Assessment of Pruritus Score. Score Grade Definition 0None No pruritus 1 Mild Clearly present but minimal awareness;occasional, slight itch 2 Moderate Definite awareness that is bothersomebut tolerable; does not disturb sleep 3 Severe Hard to tolerate;interferes with sleep

Example 26 Treatment of Test Persons Suffering from Atopic Dermatitiswith QβG10 and Assessment of Efficacy

Treatment with QβG10: A double-blind parallel-group clinical trial isperformed with 36 patients with atopic dermatitis. Patients areallocated randomly to two groups of 18 patients each. The first group istreated with 300 μg QβG10 (Qβ VLP filled with G10 (SEQ ID NO:27)) sixtimes in intervals of 1 week. The second group is treated with placebo.

Assessment of Efficacy: Physician Global Assessment (PGA), EASI Scores,and Patient's assessment of pruritus (see Example 25) are performedprior to the first treatment, bi-weekly during the treatment period, andseveral times after treatment until the end of the study at 24 weeksafter the first treatment. The changes in the symptom scores from beforethe treatment are compared between the groups at the various assessmentpoints after the treatment.

Example 27 Treatment of Test Persons Suffering from Asthma with QβG10 orAP205G10 and Assessment of Efficacy Using a Standardized Asthma Qualityof Life Questionnaire

27.1 Assessment of Asthma Symptoms: Asthma symptoms are assessed usingthe standardized Asthma Quality of Life Questionnaire (AQLQ(S) orAQLQ12+, Juniper et al., Health Qual. Life Outcomes September 20053:58).

27.2 Treatment with QβB10 or AP205G10: Two test groups and one controlgroup of persons suffering from asthma and showing comparable symptomscore with AQLQ(S) or AQLQ12+ are treated with VLPs of bacteriophage Qβpackaged with G10 (SEQ ID NO:27), VLPs of bacteriophage AP205 packagedwith G10 (SEQ ID NO:27) or placebo, respectively. QβG10 and AP205G10 areproduced according to Examples 16 and 17 of WO03/024481 or preferablyaccording to the Examples herein. The test persons are treated at leastthree times with 300 μg QβG10 or 300 μg AP205G10 in intervals of 1 week.Dependent on the average symptom score of the test group the treatmentof the test group can be repeated up to a total of 6 to 10 vaccinationswith 300 μg VLP-G10 each; the control group is always treated withplacebo in parallel.

27.3 Assessment of Efficacy: All test persons are scored using theAQLQ(S) or AQLQ12+ method directly before each treatment and 1 and 3months after the last treatment. The development of the average score inthe two groups treated with CpG packaged VLPs and the control group arecompared.

Example 28 Assessment of Asthma Symptoms

The severity of asthma symptoms can be assessed by spirometricmeasurements before and after administration of methacholine accordingto the following protocol.

Step 1: Preparation of test solution

-   -   Remove the test solution (methacholine) from the refrigerator 20        minutes before testing and bring it to room temperature;    -   Check the expiry date of the test solution;        Step 2: Preparation of test patient    -   Let patient adapt to room climate for 10 min;    -   Perform the respiratory spirometer test (see Appendix 11.4        Respiratory Spirometry);    -   Exclude contraindications for methacholine testing (relevant        airway obstruction, acute infection, pregnancy, β-blocker or        anti-cholinergic medication) (cholinergic or anticholinergic);

Step 3: Preparation of “Dosimeter Mefar MB3”

-   -   Switch on machine by pressing the two yellow buttons (“unit”,        “compressor”) on the right and wait till the pressure reaches 25        kg/cm²;    -   define inhalation time by pressing the grey numbered buttons 05,        then define the time of pause 050 and inhalation time (N.        inhale) 02, press “Reset”    -   Prepare the inhalation set by filling in lower plastic piece of        the inhalation set 1.5 ml of NaCl 0.9%, screw on upper part with        the same number, then put on linking and mouth piece and hose        that connects the inhalation instrument with the machine    -   Press the button for “Air Tank Release”        Step 4: Negative control and instruction of the patient    -   Perform negative control with NaCl 0.9% by asking the patient to        enclose the mouth piece with his lips and take a deep breath.        While taking a deep breath press button;    -   “Emergency Manual Thermistor”;    -   Repeat once (see dose protocol below), then press “End” and        “Reset 02”;    -   Wait for 2 minutes, then repeat the respiratory spirometer test;        Step 5: Provocation with methacholine    -   Fill in lower piece of inhalation set 1.5 ml of methacholine        concentration 10 mg/ml and screw set back together and press        “Air Tank Release”;    -   Ask the patient to enclose the mouth piece with his lips and to        take a deep breath. While taking a deep breath press button        “Emergency Manual Thermistor”;    -   Repeat once (see dose protocol below, Table 25). then press        “End” and “Reset 02”;    -   Wait for 2 minutes, then repeat the respiratory spirometer test;

TABLE 25 Dosage protocol for methacholine challenge. Methacholine Numberof Dose per Total Cumulative concentration inhalations inhalation dosedose NaCl 0.9% 2 — — — 10 mg/ml 2  50 μg 100 μg  100 μg 10 mg/ml 3  50μg 150 μg  250 μg 50 mg/ml 1 250 μg 250 μg  500 μg 50 mg/ml 2 250 μg 500μg 1000 μg 50 mg/ml 4 250 μg 1000 μg  2000 μg

Flowchart for the Respiratory Spirometry (RS) Step 1: Preparation

-   -   Calibrate the spirometer daily before use

Step 2: Test

-   -   Ask the subject to sit in an upright position, breathing        normally;    -   Instruct the subject to inhale as much as possible, and then to        exhale into the spirometer as rapidly and completely as        possible, until all air is exhaled;    -   Repeat two more times (total 3 times):

Step 3: Result

-   -   Define the best effort (defined as the greatest FEV₁/FVC ratio).        If the greatest FEV₁/FVC ratio is seen in more than one effort,        the best effort will be the one with the highest FEV₁;    -   If the greatest FEV₁/FVC ratio is LESS than 70% of normal        categorize as “relevant airway obstruction” and prove exclusion        criteria. Otherwise continue;    -   Subtract 20% off of measured FEV₁ and define it as FEV₁-20%;    -   Transcribe the measured FEV₁, the “FEV₁-normal” and the        calculated FEV₁-20% onto prepared sheets        (“Methacholine-(Broncho)Provocation Test”);

Example 29 Treatment of Test Persons Suffering from Asthma with QβG10 orAP205G10 and Assessment of Efficacy Using Spirometry after MethacholineChallenge

29.1 Assessment of Asthma Symptoms: Asthma symptoms are assessed bySpirometry with measurements obtained before and after theadministration of methacholine according to Example 28. Determined arethe parameters FEV1 and the ratio of FEV1 to the forced vital capacityPVC expressed as a percentage of the predicted value. Additionally, thetest persons (or their parents or guardians) complete a diary card eachday throughout the study recording night awakenings due to asthma,morning and evening peak flows as measured by peak-flow meters, use ofmedication, use of rescue medicine (albuterol) for symptoms and toprevent exercise-induced bronchospasm, use of prednisone, absence fromschool/work due to asthma, visits to a physician's office or hospitalbecause of asthma and severity of symptoms. Test persons use their usualmedication throughout the study.

29.2 Treatment with QβG10 or AP205G10: see Example 27.2.

29.3 Assessment of Efficacy; Spiromenty data of all test persons areobtained directly before each treatment, diary data are obtained 1 monthbefore the first treatment until 3 month after the last treatment on adaily basis. Analyzed are, for example, the number of asthma relatednight awakenings per month, the number of episode-free days and themorning peak flow. Average data of the two groups treated with CpGpackaged VLPs are compared to the placebo group.

Example 30 Skin Prick Test

A skin prick test (SPT) is performed to verify that a subject showshypersensitization to a certain allergen. The test is useful to test thereaction of a patient to a wide range of allergens, including pollenallergen and allergen mix that is routinely used to screen for atopy.The test is useful to test the reaction towards dust mite allergens (Derp and Der f). At Screening this test must be performed prior to CPT(Example 31). Skin prick tests and intradermal skin tests allow avisualisation of sensitisation. The basic principle of skin testing isthe introduction of a small amount of allergen into the dermis. Thereleased mediators cause vasodilatation and increase vascularpermeability, which in turn leads to tissue edema and the development ofa weal. Histamine triggers the release of the neuromediator substance Pby an axon reflex, which causes the surrounding skin to flush.

A small drop of about 30 μl of serial dilutions of the allergen isplaced on the volar surface of the forearm. The test sites must be atleast 2 cm apart to avoid false-positive reactions. Using a so calledprick needle, i.e. a needle that can be perpendicularly inserted intothe skin, the allergen solution is “pricked” into the dermis, givinghighly reproducible results. For a negative control, the diluent of theallergen solution is used. Histamine is used as a positive control todetect suppression of cutaneous reactivity by medications (mainlyantihistaminic drugs). A histamine solution at 1 mg/ml induces a wealranging from 2-7 mm in diameter.

Areas of weal and flare reactions will be recorded after 15 min. Toobtain a permanent record, the size of the wheal as well as the flare isoutlined on the skin with a pen, then blotted onto a cellophane tape andstored on paper. Both the area of the wheal and the flare will beassessed by planimetry. Therefore the reactions are scanned by acomputer and the surface areas are measured using commercial software. Aweal size of 7 mm2 or greater is regarded as positive.

Knowing that inter-individual variation in the skin response tohistamine is considerable, the reaction will then be scored as apercentage of the positive control. The evaluation of the end-pointtitrations will be based on parallel line bioassay and on median slope.

Flowchart for Skin Prick Test (SPT) Step 1: Preparation

-   -   Ask Patient about recently taken medication (corticosteroids,        anti-allergic therapy, neuroleptical and antidepressant therapy)        and exclude contraindications for SPT;    -   Check test solutions (expiry date, control temperature, body        temperature required);    -   Clean test site (volar side of arm) with alcohol. Use a pen to        mark those areas of the arm where allergens are to be pricked.        These prick sites should be at least 2 cm apart.;    -   The concentrations to be used for dust mites will be 1:1000,        1:100, 1:10 and 1:1:

Step 2: Test

-   -   A drop of the allergen solution is placed onto each of the        marked areas of the skin. A sterile prick lancet with 1 mm point        is used to prick the drop into the skin. This should not cause        any bleeding. The lanced is wiped with an alcohol swab between        pricks, in order to prevent carry-over of allergens;    -   The house dust mite allergen tests will be performed with        allergen concentrations of 1:1000, 1:100, 1:10 and 1:1;    -   A positive (histamine) and negative control (diluent) must be        included;    -   After 1 minute the solutions must be blotted, not wiped, off the        test site;    -   Wait for 30 minutes;

Step 3: Result

-   -   Wipe test site with alcohol, then with wound benzine, draw a        line around the erythema and edema with a marker pen;    -   Stick clear scotch tape over test site, then rip it off and        stick it onto a paper sheet;    -   Determine the diameter and/or the area of the erythema and        edema. Record the measurements in the source document and        transcribe onto the case report form;    -   Itching test sites can be treated topically with anti-histamine        gel;

The allergic reaction is always assessed with respect to the controlreaction (diluent only). Typically. subjects are considered allergic ifthey exhibit at least one edema or erythema of at least 6 mm diameter orat least one edema or erythema with an area of at least 7 mm².

Example 31 Conjunctival Provocation Test

The response of a subject to an allergen can be assessed using the socalled conjunctival provocation test which is performed according to thefollowing procedure. The test is useful to test the reaction of apatient to a wide range of allergens, including pollen allergen. Thetest is useful to test the reaction towards dust mite allergens (Der pand Der f).

Flowchart for the Conjuctival Provocation Test (CPT); Step 1:Preparation

-   -   Let patient adapt to room climate for 10 min;    -   Check test solutions (expiry date, control temperature, body        temperature required);    -   Confirm that the eye is without irritation at the beginning of        the provocation;    -   Exclude contraindications for CPT (any eye disease except for        anomalies of refraction or allergic conjunctivitis, contact        lenses, anti-allergic therapy);

Step 2: Control

-   -   Administer 50 μl of control solution identical to the allergen        solution except for allergen content in the lower conjunctival        sac of left eye (control eye);        Step 3: Provocation with allergen concentration 1    -   Immediately after application of control solution, administer 50        μl of allergen solution concentration 1 in the lower        conjunctival sac of the right/opposite eye (provocation eye);    -   Inform the patient not to rub his/her eye;    -   Wait for 10 min, then fill out the two symptom scores for CPT;        Step 4: Response to provocation with allergen    -   If positive, administer topical antihistamine and stop CPT;    -   If negative, repeat provocation with the next higher allergen        concentration in provocation eye until concentration 4 (see step        5);    -   If negative after provocation with concentration 4, categorize        as negative and stop;        Step 5: Provocation with next higher allergen concentration    -   administer 50 μl of allergen solution with next higher        concentration/number in the lower conjunctival sac of the        opposite eye (provocation eye);    -   Inform the patient not to rub his/her eye;    -   Wait for 10 min. then fill out the two symptom scores for CPT        (see step 4):

Categorization of the Response to Allergen in the CPT (Stage Criteria)

0: no subjective or visible reaction;I: itching, reddening, foreign body sensation;II: stage I and in addition tearing, vasodilatation of conjunctivabulbi;III: stage 11 and in addition vasodilation and erythema of conjunctivatarsi, blepharospasm;IV: stage III and in addition chemosis, lid swelling;

The stages are determined for the following solutions (dilution factorof the standard allergen solution): Negative Solution; Concentration I:1:1000, Concentration II: 1:100; Concentration III: 1:10; andConcentration IV: 1; The CPT is positive if the response is stage II orhigher.

Symptoms are scored using the Score Sheet for CPT (Table26) whichprovides for scores from 0 to 3 for 5 different parameters. Thus, themaximum score is 15 per challenge. The CPT is positive if the totalresponse is >10.

TABLE 26 CPT Score Sheet—Challenge Symptom Questionnaire. Symptom NoneMild (1) Moderate (2) Severe (3) 10' Score Conjunctival none SlightDefinite severe hyperemia redness redness redness Tearing none SlightDefinite need to sensation sensation whipe off Itching none SlightDefinite need to sensation sensation rub eyes Burning none SlightDefinite severe sensation sensation sensation Swelling of none SlightDefinite unable to eyelids sensation sensation open eyes

Example 32 Nasal Provocation Test

Nasal provocation test is useful to test the reaction of a patient to awide range of allergens, especially pollen allergens. The nose providesan ideal site for allergen provocation. Allergen can be applied to themucosa with a high degree of accuracy. The challenge procedure shouldreflect natural exposure. Quantitative measurements with highreproducibility are important (Andersson M, L. Greiff, C. Svensson andC. Persson. Acta Otolaryngol. 115 (1995), pp. 705-713). Recently,Bousquet (Bousquet J, P. van Cauwenberge and N, Khaltaev. Aria WorkshopGroup; WHO, J. Allergy Clin. Immunol. 108 (2001), pp. S147-334.) and theARIA Workshop Group have presented a document on allergic rhinitis andits impact on asthma (Bousquet et al., 2001), presenting guidelines froma subcommittee of the “International Committee on Objective Assessmentof the Nasal Airways” for nasal provocation tests concerningindications, techniques, and evaluation of the tests (Malm L, R. Gerthvan Wijk and C. Bachert. Rhinology 38 (2000), pp. 1-6.). Also, theGerman Society for Allergology and Clinical Immunology worked out aposition paper together with the Working Group for Clinical Immunology,Allergology and Environemental Medicine of the German Society for Ear,Nose and Throat (Reichelmann et al. Position statement. Allergo J (2002)11:29-36.).

Deposition of the allergen and allergen dosing: The solutions ofallergen will be delivered from a meter-dose pump spray, as the nasalpump spray delivers the solution over a large area of the nasal mucosa.The allergen solution will be an isotonic, buffered aqueous solutionwith a neutral pH. Serial 1:3 dilutions with saline will be freshlyprepared prior to testing. The exact allergen concentrations are to bedetermined in a previous study. The allergen solution will be applied ata volume of 100 μl.

Contraindications for NPT: Episode of rhinitis in last 4 weeks;Exacerbation of allergic disease; Use of allergen known to have causedanaphylactic reaction; Pregnancy: Nasal surgery in last 8 weeks;Coexisting severe general disease, especially cardiopulmonary diseases;Treatment with medications that may interfere with the treatment ofsystemic allergic reactions (e.g. β-blockers or ACE inhibitors);Vaccinations within one week prior to testing.

Withdrawal period for medications known to interfere with nasalprovocation testing: Antihistamines, systemic: 48 hours to 1 weekdepending on their half life; Antihistmines, nasal: 1 week; Ketotifen: 2weeks; DNCG, Nedocromil: 3 days; Corticosteroids, nasal: 1 week;Corticosteroids, systemic 1 week; Topical β-adrenergic agonists: 1 day;Nonsteroidal antiinflammatory drugs (NSAIDs): 1 week; Antihypertensives(e.g. reserpine, clonidine): 3 weeks; Antidepressants (e.g.,imipramines, tricyclics): 3 days.

Technique and Practical Protocol of Nasal Provocation Testing

Rhinoscopic examination including anterior or posterior rhinoscopy,preferably anterior rhinoscopy, will be performed preceding NPT in orderto evaluate the baseline condition. Patients should be well adapted toroom temperature for at least 15 minutes before a baseline evaluation byrhinoscopy and a clinical symptom score, as well as rhinomanometry. Thewider side of the nose is used for the challenge. Before administrationof the actual allergen solution, the nasal mucosa of the wider side ofthe nose is challenged for unspecific hyperresponsiveness by 100 μl ofthe allergens diluent. Evaluation of the scores and rhinomanometry isrepeated after 10 minute; to check on clinical symptoms or significantchanges in objective measurements of nasal patency. If no significantchanges occur, the actual allergen provocation is performed on the widerside of the nose. The applicator of the delivery device is inserted intothe nasal vestibulum and pointed upward and laterally towards the medialcanthus of the eye to deposit allergen on the inferior and the middleturbinate mucosa when spraying 100 μL of allergen test solution into thenose. During allergen application, the patient must hold his or herbreath to avoid inhaling the allergen into the lower airways. Patientsare told to inhale before and to exhale right after the application.

Measurements of Response

Nasal symptom recording will be performed in parallel according to threecommon methods (Bachert C: Nasal provocation test: critical evaluation.In Ring J, Behrendt H D, editors: New trends in allergy, IV, Berlin,1997, Vieluf Springer-Verlag, p 277).

Score 1: Severity of each nasal symptom is recorded on a 10-cm linearvisual analog scale. The severity is then evaluated based on the score(mild 1-3 cm; moderate 4-7 cm; severe 8-10 cm) obtained with diluent(negative control) and each provocation dose.

Score 2: A practical scoring system for a standardized quantification ofthese clinical parameters, which has been proposed by the ENT section ofthe German Society for Allergology and Clinical Immunology is summarizedin Table27 (Reichelmann et al. Position statement. Allergo J (2002)11:29-36.).

TABLE 27 Scoring system for evaluation of clinical symptoms after nasalprovocation (“Score 2”). Symptom Severity Score (points) Secretion Nosecretion 0 Little secretion 1 Heavy secretion 2 Irritation 0-2 sneezes0 3-5 sneezes 1 >5 sneezes 2 Extranasal None 0 symptoms Tearing/itching1 Conjunctivitis/chemosis + 2 Urticaria + Coughing/dyspnoea

Score 3: This score is often used in both clinical and scientificresearch studies (Linder, 1988). The end point is considered the amountof stimulant that produces a total symptom score of 5 from a maximumscore of 13 points (Table 28).

TABLE 28 Scoring system for evaluation of clinical symptoms after nasalprovocation (“Score 3”). Nasal symptoms Point score Sneezing 0 to 2sneezes 0 3 or 4 sneezes 1 5 or more sneezes 3 Pruritus Nose 1 Palate 1Ear 1 Rhinorrhea 0 to 3 Nasal Blockage 0 to 3 Ocular Symptoms 1Rhinorrhea: 0 = mild, 1 = moderate, 3 = severe; sneezing: 0 = ≦2sneezes; 1 point = 3-5 sneezes; 2 points = >5 sneezes). Other symptomsinclude itching or tearing (1 point) and conjunctivitis, cough,urticaria, or dyspnea (2 points).

Rhinorrhea: 0=mild., 1=moderate, 3=severe; sneezing: 0=0-2 sneezes; 1point 3-5 sneezes; 2 points=>5 sneezes). Other symptoms include itchingor tearing (1 point) and conjunctivitis, cough, urticaria, or dyspnea (2points).

End Point: After the allergen challenge the end point is reached whenthe patient has more than 3 points in score 2, or if the reduction innasal flow rate is >40% at 150 Pa. The end point shall also be reached,if the reduction in nasal flow rate is >20% at 150 Pa in conjunctionwith >2 points in score 2.

Objective measurement of nasal patency: Nasal air-space volume andpatency will be assessed by anterior rhinomanometry. It is designed toanalyze the transnasal airflow in one nostril at a time depending ontransnasal pressure. which is assessed contralaterally during tidalbreathing. For complete and state of the art evaluation of nasalpatency, this method is combined with acoustic rhinometry, whichdocuments nasal geometry as cross-sectional area within the nasalcavity.

Results of both methods, anterior rhinomanometry and acousticrhinometry, are visualized graphically to facilitate analysis anddocumentation. Measurements before and after NPT can be easily comparedand accurately assessed using these graphs in combination with the exactnumbers for nasal airflow (cm3/s) and cross-sectional area (cm2), whichare computed by the respective analyzers.

Example 33 Treatment of a Test Person Suffering from Pollen Allergy andAtopic Eczema with QβG10 and Assessment of Efficacy Using a NasalProvocation Test

An individual suffering from pollen allergy and atopic eczema wastreated four times by subcutaneous injection of 300 μg QβG10 in 1 weekintervals. A skin prick test according to Example 30 and nasalprovocation test according to Example 32 were performed on the daybefore the first treated to determine a baseline. The nasal provocationtest was performed with a commercial challenging agent containing pollenallergen in dilutions of 1:1000, 1:100, 1:10 and 1:1 and repeated 1 weekafter the second treatment (directly before the third treatment) and 1week after the fourth treatment. The reaction of the test person wasscored using score 2 and score 3 as described in Example 32.Additionally, the number of sneezes was recorded (Table 31). As shown inTables 29-31 all three parameters indicate a significant reduction ofthe test person's reaction to allergen challenge. Additionally, the testperson who is typically suffering from atopic eczema, with symptomsdeveloping in early winter, reported to be totally free of symptoms ofatopic eczema after the study, which was performed in late autumn. Thelatter observation is an indication for a prophylactic effect of thetreatment with respect to atopic eczema.

TABLE 29 Scoring result after nasal provocation test (Score 2). 1:10001:100 1:10 1:1 baseline 1 2 6 4 1 week after 2nd treatment 0 0 1 6 1week after 3rd treatment 0 0 0 1

TABLE 30 Scoring result after nasal provocation test (Score 3). 1:10001:100 1:10 1:1 baseline 2 7 9 9 1 week after 2nd treatment 0 1 4 9 1week after 3rd treatment 0 0 0 2

TABLE 31 Number of sneezes after nasal provocation test. 1:1000 1:1001:10 1:1 baseline 1 2 6 1 1 week after 2nd treatment 0 0 1 7 1 weekafter 3rd treatment 0 0 0 0

Example 34 Treatment of Test Persons Suffering from Pollen Allergy withQβG10 and QβG8-8 and Assessment of Efficacy Using a Nasal ProvocationTest

34.1 Treatment with QβG10 QβG8-8: After determination of a base linewith skin prick and nasal provocation test (see Examples 30 and 32)three groups of test persons suffering from pollen allergy are formed,wherein all groups show a similar test score in average. The first groupis treated at least three times in intervals of 1 week with 300 μg QβG10(Qβ VLP packaged with G10 (SEQ ID NO:27)). The second group is treatedin parallel with 300 μg Qβ packaged with G8-8 (SEQ ID NO 25). The thirdgroup is treated with placebo. Dependent on the average symptom score ofthe test group during the study the treatment of the test group can berepeated up to a total of 6 to 10 vaccinations with 300 μg of QβG10 orQβGS-8, respectively: the control group is always treated with placeboin parallel.

34.2 Assessment of Efficacy: Nasal provocation test is performed beforeeach treatment and in intervals up to 3 months after the last treatment.The changes in the symptom scores of the test group at the beginning andat the end of the study are compared to that of the control group.

Example 35 Treatment of Test Persons Suffering from House Dust Allergywith QβG10 and QβG8-8 and Assessment of Efficacy Using the ConjunctivalProvocation Test

35.1 Treatment with QβG10 QβG8-8: After determination of a base linewith skin prick and conjunctival provication test (see Examples 30 and31) three groups of test persons suffering from house dust allergy areformed, wherein all groups show a similar test score in average. Thefirst group is treated at least three times in intervals of 1 week with300 μg QβG10 (Qβ VLP packaged with G10 (SEQ ID NO:27)). The second groupis treated in parallel with 300 μg Qβ packaged with G8-8 (SEQ ID NO 25).The third group is treated with placebo. Dependent on the averagesymptom score of the test group during the study the treatment of thetest group can be repeated up to a total of 6 to 10 vaccinations with300 μg of QβG10 or QβG8-8, respectively; the control group is alwaystreated with placebo in parallel.

35.2 Assessment of Efficacy: Conjunctival provocation test is performedbefore each treatment and in intervals up to 3 months after the lasttreatment. The changes in the symptom scores of the test group at thebeginning and at the end of the study are compared to that of thecontrol group.

Example 36 Treatment of Test Persons Suffering from House Dust Allergywith HBc and HBcG8-8 and Assessment of Efficacy Using the ConjunctivalProvocation Test

36.1 Treatment with HBcG10 HBcG8-8: After determination of a base linewith skin prick and conjunctival provication test (see Examples 30 and31) three groups of test persons suffering from house dust allergy areformed, wherein all groups show a similar test score in average. Thefirst group is treated at least three times in intervals of 1 week with300 μg HBcG10 (HBc VLP packaged with G10 (SEQ ID NO:27)). The secondgroup is treated in parallel with 300 μg HBc packaged with G8-8 (SEQ IDNO 25). The third group is treated with placebo. Dependent on theaverage symptom score of the test group during the study the treatmentof the test group can be repeated up to a total of 6 to 10 vaccinationswith 300 μg of HBcG10 or HBcG8-8, respectively; the control group isalways treated with placebo in parallel.

36.2 Assessment of Efficacy: Conjunctival provocation test is performedbefore each treatment and in intervals up to 3 months after the lasttreatment. The changes in the symptom scores of the test group at thebeginning and at the end of the study are compared to that of thecontrol group.

Example 37 Treatment of Test Persons Suffering from Pollen Allergy withQβG10 and Assessment of Efficacy Using a Nasal Provocation Test, SkinPrick Test, and Patient Diary

Treatment with QβG10: An open-label clinical trial was performed withpatients with allergy to grass pollen. Patients were treated with 300 μgQβG10 (Qβ VLP filled with G10 (SEQ ID NO:27)) six times in intervals of1 week.

Assessment of Efficacy: Nasal provocation test with a standardized grasspollen extract (see Example 32) was performed prior to the firsttreatment and 2 weeks after the last treatment. Nasal provocationtesting is also performed at months 6 and 12 following the firsttreatment. The changes in the symptom scores from before the treatmentare assessed at the various assessment points after the treatment.Efficacy is also measured with the skin prick test (see Example 30)using solutions containing various amounts of grass pollen allergens.Efficacy of the treatment in daily life is measured using a validatedpatient diary to record symptoms and medication use during the firstpollen season following the treatment. The validated patient diary isalso used to record symptoms and medication use during the second pollenseason following the treatment.

Results of the nasal provocation test: 5 patients with grass pollenallergy treated with 6 weekly injections of 300 μg QβG10 were subjectedto nasal provocation (Example 32) before treatment and 2 weeks after thetreatment. As shown in Table 32 these patients showed reduced symptomscore as compared to their reaction before the treatment.

TABLE 32 Symptoms due to nasal provocation were assessed using scoresystem 2 (Example 32). Before Treatment After Treatment Allergensolution (dilution) Allergen solution (dilution) Con- 1/ 1/ 1/ 1/ Con-1/ 1/ 1/ 1/ Patient trol 1000 100 10 1 trol 1000 100 10 1 A 0 0 0 0 4 00 0 0 1 B 0 0 3 2 5 0 0 0 0 1 C 0 0 1 0 4 0 0 0 0 2 D 0 0 1 2 3 0 0 0 01 E 0 nd nd nd 5 0 0 0 2 5 F 1 1 1 1 4 0 0 0 0 1 G 0 1 1 2 4 0 0 1 1 4 H0 0 1 1 3 0 0 0 0 1 I 0 0 0 0 3 0 0 0 0 1 J 1 1 2 5 nd 0 0 0 0 2 nd =not determined

Example 38 Treatment of Test Persons Suffering from Pollen Allergy withQβG10 and Assessment of Efficacy Using a Conjunctival Provocation Test,Skin Prick Test, and Patient Diary

Treatment with QβG10: A double-blind parallel-group clinical trial isperformed with 30 patients with allergy to grass pollen. Patients areallocated randomly to three groups of 10 patients each. The first groupis treated with 300 μg QβG10 (Qβ VLP packaged with G10 (SEQ ID NO:27))six times in intervals of 1 week. The second group is treated with 300μg QβG10 in combination with the adjuvant aluminum hydroxide six timesin intervals of 1 week. The third group is treated with placebo.

Assessment of Efficacy: Conjunctival provocation test with astandardized grass pollen extract (see Example 31) is performed prior tothe first treatment, 2 weeks after the last treatment, and 6 and 12months after the first treatment. The changes in the symptom scores frombefore the treatment are compared between the groups at the variousassessment points after the treatment. Efficacy is also measured withthe skin prick test (see Example 30) using solutions containing variousamounts of grass pollen allergens. Efficacy of the treatment in dailylife is measured using a validated patient diary to record symptoms andmedication use during the first pollen season following the treatment.The validated patient diary is also used to record symptoms andmedication use during the second pollen season following the treatment.

Example 39 Treatment of Test Persons Suffering from House Dust MiteAllergy with QβG10 and Assessment of Efficacy Using ConjunctivalProvocation Test, Skin Prick Test, and Patient Diary

Treatment with QβG10: A double-blind parallel-group clinical trial isperformed with 20 patients with allergy to house dust mites. Patientsare allocated randomly to two groups of 10 patients each. The firstgroup is treated with 300 μg QβG10 (Qβ VLP packaged with G10 (SEQ IDNO:27)) in combination with the adjuvant aluminum hydroxide six times inintervals of 1 week. The second group is treated with placebo.

Assessment of Efficacy: Conjunctival provocation test with astandardized house dust mite extract (see Example 31) is performed priorto the first treatment, 2 weeks after the last treatment, and 6 and 12months after the first treatment. The changes in the symptom scores frombefore the treatment are compared between the groups at the variousassessment points after the treatment. Efficacy is also measured withthe skin prick test (see Example 30) using solutions containing variousamounts of house dust mite allergens. Efficacy of the treatment in dailylife is measured using a validated patient diary to record symptoms andmedication use during 14 consecutive days prior to treatment, two weeksafter treatment, and 6 and 12 months after the first treatment.

1. A method of treating hypersensitivity in an animal, wherein saidhypersensitivity is allergy or asthma, wherein said method comprisesintroducing a composition into said animal, and wherein said compositioncomprises: (a) a virus-like particle of an RNA bacteriophage; and (b) anunmethylated CpG-containing oligonucleotide; wherein said virus-likeparticle of an RNA bacteriophage is packaged with said unmethylatedCpG-containing oligonucleotide, and wherein said method does notcomprise co-administering an allergen to said animal, and whereinintroduction of said composition treats said hypersensitivity in saidanimal. 2-6. (canceled)
 7. The method of claim 1, wherein saidvirus-like particle of an RNA bacteriophage comprises coat proteins, orfragments thereof, of an RNA bacteriophage.
 8. The method of claim 1,wherein said RNA bacteriophage is selected from the group consisting of:(a) bacteriophage Qβ; (b) bacteriophage R17; (c) bacteriophage fr; (d)bacteriophage GA; (e) bacteriophage SP; (f) bacteriophage MS2; (g)bacteriophage M11; (h) bacteriophage MX1; (i) bacteriophage NL95; (j)bacteriophage f2; (k) bacteriophage PP7; and (l) bacteriophage AP205.9-19. (canceled)
 20. The method of claim 1, wherein said unmethylatedCpG containing oligonucleotide is capable of stimulating IFN-alphaproduction in a cell. 21-24. (canceled)
 25. The method of claim 1,wherein said unmethylated CpG-containing oligonucleotide comprises apalindromic sequence.
 26. The method of claim 1, wherein the CpG motifof said unmethylated CpG-containing oligonucleotide is part of apalindromic sequence.
 27. The method of claim 26, wherein saidpalindromic sequence is GACGATCGTC (SEQ ID NO:28).
 28. The method ofclaim 25, wherein said palindromic sequence is flanked at its5′-terminus and at its 3′-terminus by guanosine entities.
 29. The methodof claim 25, wherein said palindromic sequence is flanked at its5′-terminus by at least 3 and at most 15 guanosine entities, and whereinsaid palindromic sequence is flanked at its 3′-terminus by at least 3and at most 15 guanosine entities.
 30. The method of claim 1, whereinsaid unmethylated CpG-containing oligonucleotide comprises the sequenceselected from the group consisting of: (SEQ ID NO: 32) (a) “G6-6”GGGGGGGACGATCGTCGGGGGG; (SEQ ID NO: 33) (b) “G7-7”GGGGGGGGACGATCGTCGGGGGGG;  (SEQ ID NO: 25) (c) “G8-8”GGGGGGGGGACGATCGTCGGGGGGGG; (SEQ ID NO: 26) (d) “G9-9”GGGGGGGGGGACGATCGTCGGGGGGGGG; and (SEQ ID NO: 27) (e) “G10”GGGGGGGGGGGACGATCGTCGGGGGGGGGG.


31. The method of claim 1, wherein said unmethylated CpG-containingoligonucleotide comprises the sequence (SEQ ID NO: 27)GGGGGGGGGG GACGATCGTC GGGGGGGGGG. 


32. The method of claim 1, wherein said unmethylated CpG-containingoligonucleotide consists exclusively of phosphodiester boundnucleotides. 33-39. (canceled)
 40. The method of claim 1, wherein saidhypersensitivity is asthma.
 41. The method of claim 40, wherein saidasthma is IgE-mediated asthma. 42-53. (canceled)
 54. The method of claim1, wherein said animal is a human.
 55. (canceled)
 56. (canceled)
 57. Themethod of claim 1, wherein said method does not comprise introducing tosaid animal.
 58. (canceled)
 59. (canceled)
 60. The method of claim 1wherein an allergen is not introduced in said animal for at least oneweek before and at least one week after said introduction of saidcomposition in said animal.
 61. (canceled)
 62. The method of claim 1,wherein an allergen is not introduced in said animal for at least eightweeks before and at least eights weeks after said introduction of saidcomposition in said animal. 63-79. (canceled)
 80. The method of claim 1,wherein said virus-like particle of an RNA bacteriophage essentiallyconsists of coat proteins having the amino acid sequence of SEQ ID NO:3.81. The method of claim 1, wherein said composition does not comprise anallergen.